Dan Grisaru

ARP, a peptide derived from the stress-associated acetylcholinesterase variant has hematopoietic growth promoting activities

BackgroundPsychological stress induces rapid and long-lasting changes in blood cell composition, implying the existence of stress-induced factors that modulate hematopoiesis. Here we report the involvement of the stress-associated “readthrough” acetylcholinesterase (AChE-R) variant, and its 26 amino acid C-terminal domain (ARP) in hematopoietic stress responses.Materials and MethodsWe studied the effects of stress, cortisol, antisense oligonucleotides to AChE, and synthetic ARP on peripheral blood cell composition and clonogenic progenitor status in mice under normal and stress conditions, and on purified CD341 cells of human origin. We employed in situ hybridization and immunocytochemical staining to monitor gene expression, and 5-bromo-2-deoxyuridine (BrdU), primary liquid cultures, and clonogenic progenitor assays to correlate AChE-R and ARP with proliferation and differentiation of hematopoietic progenitors.ResultsWe identified two putative glucocorticoid response elements in the human ACHE gene encoding AChE. In human CD341 hematopoietic progenitor cells, cortisol elevated AChE-R mRNA levels and promoted hematopoietic expansion. In mice, a small peptide crossreacting with anti-ARP antiserum appeared in serum following forced swim stress. Ex vivo, ARP was more effective than cortisol and equally as effective as stem cell factor in promoting expansion and differentiation of early hematopoietic progenitor cells into myeloid and megakaryocyte lineages.ConclusionsOur findings attribute a role to AChE-R and ARP in hematopoietic homeostasis following stress, and suggest the use of ARP in clinical settings where ex vivo expansion of progenitor cells is required.

Human osteogenesis involves differentiation-dependent increases in the morphogenically active 3' alternative splicing variant of acetylcholinesterase.

ABSTRACT The extended human acetylcholinesterase (AChE) promoter contains many binding sites for osteogenic factors, including 1,25-(OH)2 vitamin D3 and 17β-estradiol. In differentiating osteosarcoma Saos-2 cells, both of these factors enhanced transcription of the AChE mRNA variant 3′ terminated with exon 6 (E6-AChE mRNA), which encodes the catalytically and morphogenically active E6-AChE isoform. In contrast, antisense oligodeoxynucleotide suppression of E6-AChE mRNA expression increased Saos-2 proliferation in a dose- and sequence-dependent manner. The antisense mechanism of action was most likely mediated by mRNA destruction or translational arrest, as cytochemical staining revealed reduction in AChE gene expression. In vivo, we found that E6-AChE mRNA levels rose following midgestation in normally differentiating, postproliferative fetal chondrocytes but not in the osteogenically impaired chondrocytes of dwarf fetuses with thanatophoric dysplasia. Taken together, these findings suggest morphogenic involvement of E6-AChE in the proliferation-differentiation balance characteristic of human osteogenesis.

Thromboembolic complications in beta thalassemia major

Thromboembolic complications in beta-thalassemia major have seldom been reported and their association with risk factors such as left ventricular failure and postsplenectomy thrombocytosis has remained speculative. In this report we describe 4 patients with unusual thromboembolic manifestations: recurrent arterial occlusion, recurrent pulmonary thromboembolism, venous thrombosis and a fatal cerebrovascular event. Although in all patients both risk factors were present, the precise causes for their thromboembolic complications were not identified. In 1 patient, however, a marked increase in hematocrit following blood transfusion resulted, in all likelihood, in a fatal cerebrovascular infarction. We suspect that these patients (constituting 4% of our beta-thalassemic group) represent a subset of those with high susceptibility to both arterial and venous thromboembolic complications.

Comparison of adnexal torsion between pregnant and nonpregnant women

OBJECTIVE The purpose of this study was to compare clinical manifestations, treatment, and pregnancy outcome of adnexal torsion in pregnant and nonpregnant women. STUDY DESIGN We conducted a retrospective case-control study in the Departments of Gynecology at 2 tertiary centers between 1999-2008. Forty-one pregnant and 77 nonpregnant women with surgically proved adnexal torsion were assessed. RESULTS Recurrence rate of torsion was 19.5% in pregnant women and 9.1% in control subjects; 73% of pregnant women conceived through assisted reproductive technologies. Doppler blood flow was falsely normal in 61% of pregnant women and in 45% of nonpregnant women; 83.3% of pregnant women delivered at term. Laparoscopic detorsion was the main surgical procedure. CONCLUSION Presentation of adnexal torsion is similar in pregnant and nonpregnant women. Past assisted reproductive technology is an important risk factor in pregnancy. Doppler blood flow has a high false-negative rate and should not outweigh clinical suspicion. Although pregnancy outcome is favorable, the high rate of recurrence raises the issue of surgical fixation at the first episode.

Hydrolytic and Nonenzymatic Functions of Acetylcholinesterase Comodulate Hemopoietic Stress Responses

Glucocorticoid-initiated granulocytosis, excessive proliferation of granulocytes, persists after cortisol levels are lowered, suggesting the involvement of additional stress mediator(s). In this study, we report that the stress-induced acetylcholinesterase variant, AChE-R, and its cleavable, cell-penetrating C-terminal peptide, ARP, facilitate granulocytosis. In postdelivery patients, AChE-R-expressing granulocyte counts increased concomitantly with serum cortisol and AChE activity levels, yet persisted after cortisol had declined. Ex vivo, mononuclear cells of adult peripheral blood responded to synthetic ARP26 by overproduction of hemopoietically active proinflammatory cytokines (e.g., IL-6, IL-10, and TNF-α). Physiologically relevant ARP26 levels promoted AChE gene expression and induced the expansion of cultured CD34+ progenitors and granulocyte maturation more effectively than cortisol, suggesting autoregulatory prolongation of ARP effects. In vivo, transgenic mice overexpressing human AChE-R, unlike matched controls, showed enhanced expression of the myelopoietic transcription factor PU.1 and maintained a stable granulocytic state following bacterial LPS exposure. AChE-R accumulation and the consequent inflammatory consequences can thus modulate immune responses to stress stimuli.

Pheochromocytoma in pregnancy: case report and review of the literature.

Pheochromocytoma is a rare disease that may occur during pregnancy. Only a few hundred cases have been published in the literature. Manifestations include hypertension with various clinical presentations, possibly resembling those of pregnancy-induced hypertension, or pre-eclamptic toxemia. Differentiation of these conditions is not always feasible, thus creating a serious risk, because fetal and maternal morbidity and mortality are far higher with pheochromocytoma. Biochemical measurements of catecholamines and their metabolites are apparently a convenient way to establish diagnosis during pregnancy, inasmuch as interpretation of radiological evaluation is complicated by the gravid uterus, and might even be potentially dangerous due to the use of ionizing radiation. More sophisticated methods for evaluation are not always practical during pregnancy. Medical treatment aims at controlling symptoms, mandating the use of alpha- and beta-receptors blockade medication. Surgical intervention is the only possible curative method available, but the critical issue is probably to identify the exact timing during the course of pregnancy for such intervention, or the ability to control symptoms until delivery. Although malignant transformation of pheochromocytoma have been reported, it is extremely uncommon. The overall prognosis is mainly affected by early diagnosis, and multidisciplinarian management.

Improved Quality of Life with Hyperbaric Oxygen Therapy in Patients with Persistent Pelvic Radiation-induced Toxicity

AIMS We report the results of hyperbaric oxygen therapy (HBOT) used in the treatment of radiation-induced persistent side-effects after the irradiation of pelvic tumours. MATERIALS AND METHODS Between January 2001 and December 2005, 13 women (median age 60.3 years) with radiation combined proctitis/cystitis (n=6), longstanding vaginal ulcers and fistulas (n=5) and longstanding skin injuries (n=2) underwent HBOT in a multiplace chamber for a median of 27 sessions (range 16-40). The treatment schedule was HBOT 100% oxygen, at 2 absolute atmospheres, for 90 min, once a day. For radiation-induced toxicity grading we used the National Cancer Institute Common Toxicity Criteria (CTC) grading system, before and after HBOT. RESULTS Thirteen patients underwent an adequate number of HBOT sessions. The mean CTC grading score before HBOT was 3.3+/-0.75, whereas the mean CTC grading score after HBOT was 0.3+/-0.63. The scores showed a significant improvement after HBOT (P=0.001; exact Wilcoxon signed-rank test). Rectal bleeding ceased in five of six patients with proctitis and dysuria resolved in six of seven cystitis patients. Macroscopic haematuria stopped in seven of seven patients. Scar complications resolved in two of two patients. None reported HBOT-associated side-effects. CONCLUSION HBOT is apparently safe and effective in managing radiation-induced late side-effects, such as soft tissue necrosis (skin and vagina), cystitis, proctitis and fistulas.

Modified testicular expression of stress-associated "readthrough" acetylcholinesterase predicts male infertility

Male infertility is often attributed to stress. However, the protein or proteins that mediate stress‐related infertility are not yet known. Overexpression of the “readthrough” variant of acetylcholinesterase (AChE‐R) is involved in the cellular stress response in a variety of mammalian tissues. Here, we report testicular overexpression of AChE‐R in heads, but not tails, of postmeiotic spermatozoa from mice subjected to a transient psychological stress compared with age‐matched control mice. Transgenic mice overexpressing AChE‐R displayed reduced sperm counts, decreased seminal gland weight, and impaired sperm motility compared with age‐matched nontransgenic controls. AChE‐R was prominent in meiotic phase spermatocytes and in tails, but not heads, of testicular spermatozoa from AChE‐R transgenic mice. Head‐localized AChE‐R was characteristic of human sperm from fertile donors. In contrast, sperm head AChER staining was conspicuously reduced in samples from human couples for whom the cause of infertility could not be determined, similar to the pattern found in transgenic mice. These findings indicate AChE‐R involvement in impaired sperm quality, which suggests that it is a molecular marker for stress‐related infertility.

Expansion of megakaryocyte progenitors from human umbilical cord blood using a new two-step separation procedure

Cord blood (CB) transplantation is primarily performed in children, rather than in adults, due to the low number of haemopoietic progenitor cells obtained from the small volume of a single CB collection. Prolonged thrombocytopenia is a major problem following CB transplantation. Efforts are currently underway to expand the number of CB progenitor cells ex vivo, in order to enable transplantation in adults and to decrease the period of thrombocytopenia. In this study we investigated different techniques for enrichment and expansion of megakaryocyte (Mk) progenitor cells and haemopoietic stem cells from CB. CBs from 20 normal deliveries were depleted of red blood cells (RBC) by dividing each sample and testing cell separation on 3% gelatin, Hespan, Ficoll‐Paque or a two‐step 3% gelatin followed by Ficoll‐Paque separation. The two‐step procedure was found to be superior to the other methods in enrichment of the Mk progenitor cells (CFU‐Mk) (34.3‐fold), while at the same time retaining the number of myeloid and erythroid progenitors, CD34+ and CD41+ cells. In short‐term (14 d) liquid culture of non‐adherent nucleated cells isolated by gelatin and Ficoll‐Paque, a 40‐fold expansion of clonable Mk progenitor cells was obtained in the presence of thrombopoietin (r‐hu‐TPO) and stem cell factor (r‐hu‐SCF). In similar cultures of isolated CD34+ cells, a 100‐fold clonable Mk progenitor was obtained at day 14. Therefore this new technique may facilitate the ex vivo expansion of Mk progenitor cells and be adopted for future use in CB transplantation.

Blood-cell-specific acetylcholinesterase splice variations under changing stimuli

Developmental and trauma‐induced mechanism(s) that modify inflammation and immune responses in blood cells were recently found to be regulated by acetylcholine. Here, we report corresponding blood cell‐specific changes in acetylcholinesterase splice variants. Plasmon resonance and flow cytometry using acetylcholinesterase variant‐specific antibody probes, revealed a progressive increase in myeloid cell fractions expressing the apoptosis‐related acetylcholinesterase‐S variant from newborns to adult controls and post‐delivery mothers. Hematopoietic cell fractions positive for the myeloproliferative acetylcholinesterase‐R variant, were similarly high in post‐partum blood, both intracellular and on the cell surface. Moreover, intracellular acetylcholinesterase‐S protein amounts as reflected by fluorescence intensity measurements remained unchanged in myeloid cells from post‐partum mothers as compared with matched controls. Unlike brain neurons, which over‐express intracellular acetylcholinesterase‐R under stress, lymphocytes from post‐partum mothers presented increased surface acetylcholinesterase‐S and pronounced decreases in both the expression and contents of surface acetylcholinesterase‐R. Peripheral stimuli‐induced modulations in acetylcholine regulation may hence reflect blood cell lineage‐dependent acetylcholinesterase splice variations.