Anemia, Hepcidin, and Vitamin D in Healthy Preterm Infants: A Pilot Study.

OBJECTIVE: The etiology of anemia in premature neonates is multifactorial and may involve anemia of inflammation mediated by hepcidin. Hepcidin expression is suppressed by vitamin D. We aimed to investigate the interrelationship between hepcidin, anemia, and vitamin D status in preterm infants. STUDY DESIGN: Preterm infants aged 1 to 5 weeks were prospectively recruited at the neonatal intensive care unit of the Dana Dwek Children Hospital. Blood counts and serum levels of hepcidin, ferritin, iron, 25-hydroxyvitamin D [25(OH)D] and C-reactive protein (CRP) were measured and compared between anemic and nonanemic preterm infants. RESULTS: Forty-seven preterm infants (mean ± standard deviation gestational age at birth 32.8 ± 1.1 weeks, 66% males) were recruited. In total, 36% of the preterm infants were vitamin D deficient [25(OH)D < 20 ng/mL] and 15% were anemic. Hepcidin levels were significantly higher in anemic premature infants than in the nonanemic group (55.3 ± 23.9 ng/mL vs. 30.1 ± 16.3 ng/mL, respectively, p < 0.05). No differences were found in iron, ferritin, 25(OH)D, and CRP levels between anemic and nonanemic premature newborn infants. A positive correlation was found between hepcidin and ferritin (R 2 = 0.247, p = 0.02) and a negative correlation was found between 25(OH)D and CRP (R 2 = 0.1, p = 0.04). No significant correlations were found between 25(OH)D and hepcidin, iron, ferritin, or CRP. CONCLUSION: Anemia of prematurity was associated with high hepcidin serum levels. The exact mechanisms leading to anemia and the role of vitamin D warrant further investigation. KEY POINTS: · Hepcidin levels were significantly higher in anemic premature infants.. · A positive correlation was found between hepcidin and ferritin.. · Negative correlation was found between 25(OH)D and CRP..

Pathogenic heparin-induced thrombocytopenia and thrombosis (HIT) antibodies determined by rapid functional flow cytometry.

OBJECTIVES: Reliable diagnosis of heparin-induced thrombocytopenia and thrombosis (HIT) is mandatory for patient management, yet prompt determination of pathogenic antibodies remains an unmet clinical challenge. Common immunoassays carry inherent limitations and functional assays which detect antibody-mediated platelet activation are not usually readily available to routine laboratories, especially the serotonin release assay (SRA), being technically demanding, time consuming, and requires high level expertise. To overcome some of these limitations, we have developed a practical functional flow cytometric assay (FCA) for routine clinical use. METHODS: A simple FCA is described which avoids platelet manipulation, is highly specific and sensitive compared with SRA, and provides rapid results. RESULTS: Of the 650 consecutive samples, from HIT-suspected patients, 99 (15.3%) were positive by the PaGIA Heparin/PF4 immunoassay and 31 (4.8%) by FCA. Average platelet activation was 11-fold higher in PaGIA+/FCA+ vs PaGIA-/FCA- samples. Of 21 SRA-positive samples, 19 were FCA-positive (relative sensitivity 90.5%), and of 42 SRA-negative samples, 40 were FCA-negative (relative specificity 95.2%). The FCA showed significantly higher correlation with the clinical presentation of HIT (4Ts score) performed on 182 patients, compared with PaGIA Heparin/PF4 (ROC-plot analysis, AUC 0.93 vs 0.63, P < 0.001). At a 92% sensitivity, the assay specificity was 96%. CONCLUSIONS: The present FCA is practical for routine testing, providing prompt reliable results for initial diagnosis and confirmation, to effectively assist in HIT patient management.

The interrelationship between hepcidin, vitamin D, and anemia in children with acute infectious disease.

BACKGROUND: Hepcidin is a master regulator of iron metabolism. Recently, it has been shown that vitamin D suppresses hepcidin expression. Our hypothesis was that hepcidin levels inversely correlate with vitamin D levels in anemic children during acute infection. METHODS: A prospective study was performed on 90 patients (45 females, 45 males, mean age 7.3 ± 5 years) who were admitted to the pediatric ward. Sixty-two patients had infectious disease (32 with coexisting anemia, 30 without anemia), and 28 patients were hospitalized for noninfectious causes. Blood samples for IL-6, hepcidin, iron status parameters, and 25-hydroxyvitamin D (25-OHD) were obtained within 72 h after admission. RESULTS: Serum concentrations of IL-6 and hepcidin were significantly higher and 25-OHD, iron, and transferrin were significantly lower in anemic children with infectious disease compared with controls. Children with a serum 25-OHD level < 20 ng/ml had significantly increased odds of having anemia than those with a level > 20 ng/ml (OR: 6.1, CI: 1.15-32.76). Correlation analyses found positive associations between hepcidin levels and ferritin (R2 = 0.47, P < 0.001) and negative associations between hepcidin and transferrin (R2 = 0.57, P < 0.001). CONCLUSION: Higher IL-6 and lower 25-OHD levels may lead to higher hepcidin levels and subsequently to hypoferremia and anemia in children with acute infection.

International Society for Cell Therapy Facility Sanitization Survey Laboratory Practices Committee Report.

BACKGROUND AIMS: Quality cell manufacturing processes require a clean laboratory environment. METHODS: This report was aimed at describing current cleaning and sanitization practices reported by facilities that manufacture many types of cellular therapy products for clinical use. It is our hope that this report may provide the groundwork for guidance recommendations directed at developing consensus standards for cleaning and sanitization practices across the globe. Facility sanitization is a central issue to regulatory and accreditation bodies. Facilities are required to develop plans to assess sanitization practices and test cleaning effectiveness. RESULTS: This document provides information on how this is performed in different facilities and may allow newer, smaller or less developed facilities to build, enhance or revise their current quality program by using experience and expertise in facility sanitization reported herein. CONCLUSIONS: This report summarizes the results of the latest survey and compares results with those previously reported. New and relevant trends in the field provide important information and will provide important information for establishing guidelines.

Divergence in CD19-mediated signaling unfolds intraclonal diversity in chronic lymphocytic leukemia, which correlates with disease progression.

Emerging data on intraclonal diversity imply that this phenomenon may play a role in the clinical outcome of patients with chronic lymphocytic leukemia (CLL), where subsets of the CLL clone responding more robustly to external stimuli may gain a growth and survival advantage. In this study, we report intraclonal diversity resolved by responses to CD19 engagement in CLL cells, which can be classified into CD19-responsive (CD19-R) and -nonresponive subpopulations. Engagement of CD19 by anti-CD19 Ab rapidly induced cellular aggregation in the CD19-R CLL cells. The CD19-R CLL cells expressed higher surface levels of CD19 and c-myc mRNA, exhibited distinct morphological features, and were preferentially abolished in rituximab-treated patients. Both subpopulations reacted to sIgM stimulation in a similar manner and exhibited similar levels of Akt and Erk phosphorylation, pointing to functional signaling divergence within the BCR. CD19 unresponsiveness was partially reversible, where nonresponding CD19 cells spontaneously recover their signaling capacity following incubation in vitro, pointing to possible in vivo CD19-signaling attenuating mechanisms. This concept was supported by the lower CD19-R occurrence in bone marrow-derived samples compared with cells derived from the peripheral blood of the same patients. CLL patients with >15.25% of the CD19-R cell fraction had a shorter median time to treatment compared with patients with <15.25% of CD19-R cell fraction. In conclusion, divergence in CD19-mediated signaling unfolds both interpatient and intraclonal diversity in CLL. This signaling diversity is associated with physiological implications, including the location of the cells, their responses to anti-CLL therapeutics, and disease progression.

Advances in megakaryocytopoiesis and thrombopoiesis: from bench to bedside.

Megakaryocytopoiesis involves the commitment of haematopoietic stem cells, proliferation and terminal differentiation of megakaryocytic progenitors (MK-p) and maturation of megakaryocytes (MKs) to produce functional platelets. This complex process occurs in specialized niches in the bone marrow where MKs align adjacent to vascular endothelial cells, form proplatelet projections and release platelets into the circulation. Thrombopoietin (THPO, TPO) is the primary growth factor for the MK lineage and necessary at all stages of development. THPO is constitutively produced in the liver, and binds to MPL (c-Mpl) receptor on platelets and MKs. This activates a cascade of signalling molecules, which induce transcription factors to drive MK development and thrombopoiesis. Decreased turnover rate and platelet number result in increased levels of free THPO, which induces a concentration-dependent compensatory response of marrow-MKs to enhance platelet production. Newly developed thrombopoietic agents operating via MPL receptor facilitate platelet production in thrombocytopenic states, primarily immune thrombocytopenia. Other drugs are available for attenuating malignant thrombocytosis. Herein, we review the regulation of megakaryocytopoiesis and platelet production in normal and disease states, and the innovative drugs and therapeutic modalities to stimulate or decrease thrombopoiesis.

Increased CD39 expression on CD4(+) T lymphocytes has clinical and prognostic significance in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) cells depend on their microenvironment for proliferation and survival. Ectonucleotidase CD39 has anti-inflammatory properties as it hydrolyzes proinflammatory extracellular ATP, generates anti-inflammatory adenosine, and also protects regulatory T cells from ATP-induced cell death. In this study, we investigated the clinical significance of CD39 expression on CD4(+) T cells in 62 patients with CLL as well as its compartmental regulation and explored the possible mechanisms for its induction. Compared to healthy individuals, CD4(+)CD39(+) lymphocytes were increased in the peripheral blood of patients with CLL and correlated with the advanced stage of disease. CD4(+)CD39(+) cells were also higher in patients with CLL, who needed therapeutic intervention, and in those who had unmutated immunoglobulin heavy chain variable region gene, were ZAP70(+) or had ß2-microglobulin levels of >3 g/L. There were more CD4(+)CD39(+) lymphocytes in the bone marrow compartment than in the peripheral blood, and in vitro studies showed that CD39 can be induced on CD4(+) cells by exposure to ATP or indirectly, following B cell receptor engagement. This may support the notion that the leukemic cells contribute to create an immune-subversive environment, and perhaps to a poorer prognosis. CD39(+) may also serve as a future target for the development of novel therapies with immune-modulating antitumor agents in CLL.

The diagnostic yield of D-Dimer in relation to time from symptom onset in patients evaluated for venous thromboembolism in the emergency medicine department.

D-Dimer concentrations increase following the thrombotic event and decrease thereafter. Timing of D-Dimer evaluation in relation to the onset of the disease might have a diagnostic impact. We have presently performed a retrospective analysis of diagnostic procedures performed in individuals who presented to the Emergency department and evaluated for acute venous thromboembolism (VTE) following a single quantitative D-Dimer testing. Individuals who had a negative objective test served as controls to those who had a positive one (Doppler ultrasonography, high probability lung scan or a CT angiography). Seven hundred thirty-four individuals presented to the Emergency department, performed a single D-Dimer test as well as an objective test during their evaluation for an eventual event of acute VTE. One hundred ninety-seven patients had a positive objective test for either deep vein thrombosis (DVT) or pulmonary embolus. They were divided into seven tiles of times from symptoms onset. Highly significant differences between patients and controls regarding D-Dimer concentrations were noted mainly during the early days from symptom onset and turned less significant thereafter. Taking into consideration the time from symptoms onset in patients with acute VTE might have an effect on the diagnostic yield of quantitative D-Dimer in the Emergency department. We suggest not to exclude the eventual presence of acute VTE if quantitative D-Dimer is obtained later than 1 week following the onset of symptoms.

Mimicking the haematopoietic niche microenvironment provides a novel strategy for expansion of haematopoietic and megakaryocyte-progenitor cells from cord blood.

Severe neutropenia and protracted thrombocytopenia remain serious clinical problems following cord blood transplantation (CBT) due to the paucity of stem and progenitor cells in the grafts. Administration of ex-vivo expanded megakaryocyte progenitor cells may facilitate platelet production. We propose a novel strategy to expand these rare cells ex-vivo, from a small portion of the cord blood (CB) unit, using fibronectin (FN), a major component of hematopoietic niches, combined with cytokines, including thrombopoietin and the hematopoietic stress-associated acetylcholinesterase readthrough peptide (ARP). Application of multiple gates and high definition flow cytometry enabled clear resolution of expanded hematopoietic stem/precursor cells (HSPC) and megakaryocyte progenitors (Mk-p) and their early subsets while eliminating positively stained non-relevant cells. FN increased viability, expansion of all CD34(+) HSPC populations and Mk-p. The combination of FN + thrombopoietin + ARP maintained and expanded very early myeloid and thrombopoietic precursors, increased the proliferation of megakaryocyte, granulocyte-macrophage and multilineage colony-forming progenitors and supported Mk maturation as measured by ploidy and glycoprotein IIb/IIIa expression by quantiative reverse transcription polymerase chain reaction. This approach, which involves expanding HSPC and Mk precursors from a small portion of the CB unit, without sacrificing the coveted stem cells, may lead to improved cell therapy modalities to facilitate earlier myelopoiesis and platelet production post-CBT.

Recombinant human granulocyte-colony stimulating factor administration for treating amyotrophic lateral sclerosis: A pilot study.

Granulocyte-colony stimulating factor (G-CSF) is used to mobilize CD34+ haematopoietic stem cells from the bone marrow to the peripheral blood. We proposed to use cell subsets induced by G-CSF to slow down disease progression in patients with amyotrophic lateral sclerosis (ALS). Patients with definite or probable ALS were assigned in a double-blind manner to receive G-CSF or placebo every three months for a year. The primary outcome measure was the functional decline, measured by the revised ALS Functional Rating Scale, Revised (ALSFRS-R) score. Secondary outcome measures included vital capacity, manual muscle strength, compound muscle action potential amplitudes, neurophysiological index, and McGill single item quality of life score (QoL). Thirty-nine patients were enrolled. Seventeen patients who received G-CSF and 18 who received placebo were evaluated. G-CSF was effective in mobilizing CD34+ to blood. The outcome measures used showed no statistically significant benefit, although there was a trend of slowing disease progression following two G-CSF treatments, as shown by lower slopes of ALSFRS-R and QoL in the first six treatment months. The treatment had no major side-effects. G-CSF administration in ALS patients caused successful mobilization of autologous bone marrow cells, but was not effective in slowing down disease deterioration.

Mesenchymal stromal cells revert multiple myeloma cells to less differentiated phenotype by the combined activities of adhesive interactions and interleukin-6.

Multiple myeloma is characterized by the malignant growth of immunoglobulin producing plasma cells, predominantly in the bone marrow. The effects of primary human mesenchymal stromal cells on the differentiation phenotype of multiple myeloma cells were studied by co-culture experiments. The incubation of multiple myeloma cells with bone marrow-derived mesenchymal stromal cells resulted in significant reduction of the expression of the predominant plasma cell differentiation markers CD38 and CD138, and cell surface immunoglobulin light chain. While the down-regulation of CD138 by stromal cells was completely dependent on their adhesive interactions with the multiple myeloma cells, interleukin-6 induced specific down-regulation of CD38. Mesenchymal stromal cells or their conditioned media inhibited the growth of multiple myeloma cell line, thereby reducing the overall amounts of secreted light chains. Analysis of primary multiple myeloma bone marrow samples reveled that the expression of CD38 on multiple myeloma cells was not affected by adhesive interactions. The ex vivo propagation of primary multiple myeloma cells resulted in significant increase in their differentiation markers. Overall, the data indicate that the bone marrow-derived mesenchymal stromal cells revert multiple myeloma cells to less differentiated phenotype by the combined activities of adhesive interactions and interleukin-6.

[Megakaryocyte development and platelet production in normal and disease states].

Megakaryocytopoiesis involves the commitment of hematopoietic stem cell, the proliferation and terminal differentiation of the megakaryocytic progenitors and maturation to platelet producing megakaryocytes (MK). MKs align adjacent to bone marrow vascular endothelial cells, and form proplatelets. Platelets are released, or sheared from the MK directly into the circulation from the tips of proplatelets which protrude into the vascular lumen. The regulation of megakaryocytopoiesis is mediated through multiple hematopoietic growth factors, chemokines and cellular interactions via signal transduction pathways and integrated transcription factors. The primary physiological growth factor that regulates megakaryocytopoiesis and platelet production is thrombopoietin. Circulating Levels of thrombopoietin (TPO) induce concentration-dependent proliferation and maturation of MK progenitors by binding to the c-Mpl receptor and signaling induction. Increased concentration of free TPO resulting from decreased platelet turnover rates enables the compensatory response of marrow MKs to drive amplified platelet production. C-MpL signaling is orchestrated by a complex cascade of signaling molecules inducing the action of specific transcription factors to drive MK proliferation and maturation. Newly developed thrombopoietic agents operating via c-Mpl receptor have now been proven useful in supporting platelet production in thrombocytopenic states. In this article, the authors review the regulation of megakaryocytopoiesis and platelet production in normal and disease states, and new approaches of thrombopoietic therapy.

Vitamin D Decreases Hepcidin and Inflammatory Markers in Newly Diagnosed Inflammatory Bowel Disease Paediatric Patients: A Prospective Study.

BACKGROUND AND AIMS: The role of hepcidin in inflammatory bowel disease [IBD] in children with anaemia is poorly understood. However, it has been shown that vitamin D suppresses hepcidin expression. We aimed to assess serum hepcidin levels and the effect of vitamin D treatment on those levels in newly diagnosed IBD paediatric patients. METHODS: Eighty-five children were prospectively recruited in the Dana-Dwek Children's Hospital [40 newly diagnosed IBD, 45 healthy controls, 47% female, mean age 13.5 ± 3.4 years]. Blood samples for measurement of interleukin 6 [IL-6], C-reactive protein [CRP], hepcidin, iron parameters and 25-hydroxyvitamin D [25-(OH)-D] levels were obtained at baseline. Patients with mild-to-moderate signs and symptoms of IBD were treated with 4000 units of vitamin D daily for 2 weeks, after which the blood tests were repeated. RESULTS: Basal hepcidin, IL-6, CRP and platelet counts were significantly higher, and haemoglobin, serum iron and transferrin levels were significantly lower in the IBD children compared to controls [p < 0.001]. Eighteen patients completed 2 weeks of treatment with vitamin D. Following treatment, serum 25-(OH)-D concentrations increased by 40% [from 22.5 to 32.5 ng/mL], and serum hepcidin, CRP and ferritin levels decreased by 81%, 81% and 40% [from 33.9 to 6.7 ng/mL, from 23.9 to 4.7 mg/L, and from 27 to 16 ng/mL, respectively] [p ≤ 0.001]. CONCLUSION: Serum hepcidin levels were significantly higher in IBD paediatric patients compared to controls. Following vitamin D treatment, serum hepcidin concentration decreased significantly. These findings suggest a potential role for vitamin D in treating anaemia in IBD children. CLINICALTRIALS.GOV NUMBER: NCT03145896.

Circulating apoptotic progenitor cells: a novel biomarker in patients with acute coronary syndromes.

BACKGROUND: Progenitor CD34 cells are capable of differentiating into endothelial cells and play a role in neoangiogenesis. Circulating CD34+ cells and endothelial progenitor cells are increased in acute coronary syndrome (ACS) patients possibly because of peripheral mobilization. We tested the hypothesis that circulating apoptotic progenitors are detectable in healthy subjects and altered in ACS patients. METHODS AND RESULTS: Peripheral blood mononuclear cells were isolated by Ficoll density gradient from 53 patients with ACS undergoing coronary angiography and 27 healthy subjects. Apoptosis in progenitor CD34+ cells was assessed using the Annexin V-PE/7-AAD detection kit, and fluorescence-activated cell sorter analysis was performed with triple staining for CD34, annexin-V, and 7-AAD. The percentage of apoptotic CD34+ progenitors was determined in the 2 subject groups and correlated with clinical characteristics. The percentage of apoptotic CD34+ progenitor cells was significantly increased in patients with ACS as compared with healthy subjects and was associated with the extent of coronary stenosis by angiography. There was no significant correlation between apoptotic progenitor CD34+ cells and the other parameters that we examined (age, smoking, hypertension, hyperlipidemia, diabetes mellitus, ejection fraction, creatinine levels, or taking any of the various medications, including beta blockers, thiazides, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, calcium blockers, nitrates, or statins). CONCLUSION: We established for the first time to our knowledge an assay to detect circulating apoptotic progenitor cells using fluorescein isothiocyanate-anti-CD34 MAb, annexin V-PE, and 7-AAD and found that apoptotic CD34+ cells are increased in ACS patients and in patients with more extensive coronary artery disease. This novel assay may shed new light on the factors governing the hemostasis of progenitor CD34+ cells.

Correlated expression of D-dimer concentrations with thrombotic burden in acute pulmonary embolism.

Thrombotic burden might have an influence upon the concentration of D-dimer in patients with acute pulmonary embolism. Patients with small pulmonary embolisms may thus present with relatively low concentrations of D-dimer. The objective of this study was to assess the correlation of the concentrations of D-dimer with the pulmonary artery occlusion score (PAOS) in a cohort of patients with acute pulmonary embolism. We have presently studied the correlation between the concentrations of D-dimer and the PAOS in a group of 75 patients who presented to the Department of Emergency Medicine with a clinical picture suggestive for acute pulmonary embolism and whose pulmonary computerized tomography (CT) angiography was positive for pulmonary embolism. A significant (P < 0.001) correlation (r = 0.42) was noted between the concentration of D-dimer and the PAOS in this group of 75 patients with acute pulmonary embolism. We further divided the cohort into those patients who had a score below the median of 18 (n = 37) and those who had a score above the median (n = 38), the corresponding mean concentrations of D-dimer being 364 and 814 ng/ml, respectively, in contrast to a mean concentration of 285 ng/ml that was observed in the group of controls (n = 73). In addition, from the receiver-operated characteristic (ROC) curves that were produced for the purpose of differentiating between the presence or absence of pulmonary embolism, for those who had a low score it was not possible to differentiate between those who had or did not have a pulmonary embolism [area under the curve 0.595 as opposed to 0.835 (P < 0.001) for the group with the high score]. Patients with acute small pulmonary embolism might present with relatively low concentrations of D-dimer. These findings might have implications regarding the diagnostic yield of D-dimer in patients who are suspected of having an acute pulmonary embolism.

Malignant transformation of normal enterocytes following downregulation of Bak expression.

Bak is a pro-apoptotic gene, which plays an important role in the multi-step process of gastrointestinal tumorigenesis. We hypothesized that downregulation of Bak expression in normal enterocytes will result in a transformed phenotype. The nontumorigenic intestinal epithelial cell line (IEC18) was transfected with the vector pMV12-AS-bak (encoding anti-sense bak). Three clones, with Bak protein levels similar to those seen in colon cancer cell lines and significantly lower than those found in the parental cells, were further evaluated. The three clones proliferated faster, demonstrated anchorage-independent growth in soft agar and a higher saturation density and plating efficiency. Furthermore, when injected into nude mice, these cells generated tumors after approximately 2-3 weeks. The cells were more resistant to the induction of apoptosis by sulindac sulfide and sulindac sulfone but more sensitive to COX 2 inhibitors (celecoxib and nimesulide). The levels of p16, cyclin D1 and COX 2 were higher in the three transformed clones. In summary,downregulation of Bak expression in normal enterocytes contributes to abnormal growth and tumorigenesis. COX 2 inhibitors may serve as important agents in the prevention and treatment of CRC as they only inhibit the growth of malignant cells.

Heparanase modulates heparinoids anticoagulant activities via non-enzymatic mechanisms.

A key element for the physiological restriction of blood coagulation at the endothelial cell surface is its non-thrombogenic property, mainly attributed to cell surface heparan sulfate proteoglycans. Heparanase is an endo-beta-D-glucuronidase with specific heparan sulfate degrading activity, which is produced and stored in platelets, and is released upon their activation. We examined the effects of heparanase pro-enzyme on coagulation functions, predominantly under physiological conditions. While heparanase pro-enzyme does not directly affect coagulation protein activities, it has profound effects on heparinoid-mediated regulation of coagulation responses, apparently via mechanisms that do not involve its enzymatic activity. Heparanase pro-enzyme reverses the anti-coagulant activity of unfractionated heparin on the coagulation pathway as well as on thrombin activity. In addition, heparanase pro-enzyme abrogated the factor X inhibitory activity of low-molecular-weight heparin (LMWH). The pro-coagulant effects of the non-active heparanase were also exerted by its major functional heparin-binding peptide. Finally, the effects of heparanase on the activity of factor VII activating protease that is auto-activated by heparinoids indicated a complete antagonistic action of heparanase in this system. Altogether, heparanase pro-coagulant activities that were also demonstrated in plasma samples from patients under LMWH treatment, point to a possible use of this molecule as antagonist for heparinoid treatment.

Sustained leukocyte count during rising cortisol level.

AIMS: To follow the trend of leukocyte counts relative to the serum cortisol levels in hospitalized patients. METHODS: We retrospectively reviewed the charts of 59 hospitalized patients who had sequential blood profiles during a 2-week period. RESULTS: The study cohort was divided into two subgroups according to the categorical cortisol levels: those within the normal range (< 25 microg/dl; n = 31 patients) and those above the normal range (> or = 25 microg/dl; n = 28 patients). The baseline white blood cell counts (WBCCs) were similar in both groups. After 1 week, however, the WBCCs dropped significantly in the presence of normal cortisol levels and increased in the presence of elevated cortisol levels (p = 0.002). This pattern was not followed by the platelet counts. A significant correlation was observed between the cortisol levels and the 1 week concomitant WBCC (r = 0.33). CONCLUSION: Cortisol may be the mechanism with a positive effect on the maintenance of elevated leukocyte counts.

A novel and rapid in vivo system for testing therapeutics on human leukemias.

OBJECTIVE: To develop a novel in vivo system for rapid assessment of leukemia growth and treatment of human blood cell malignancies. MATERIALS AND METHODS: Cell lines derived from several human hematologic malignancies were introduced into chick embryos using four different methods. RESULTS: K562 cells engraft in 100% of embryos following intravascular or intra-amniotic injection. The engraftment is rapid, appearing as soon as 7 days after injection, in striking contrast to the 4 weeks and more required for engrafting severe combined immunodeficient mice with human leukemia by systemic injection. The engraftment is easily visualized in vivo as tumor nodules in the chicken chorioallantoic membrane (CAM). In addition, leukemia is consistently detected in the embryos' hematopoietic organs by polymerase chain reaction amplification of human-specific DNA sequences. Consistent engraftment was also obtained using another leukemia cell line (DAMI). Finally, we demonstrate proof of principle that this system can be used for testing the efficacy of chemotherapy agents. Dramatic and consistent regression of tumors in the CAM was induced by a single intravenous dose of doxorubicin administered to K562-engrafted embryos. CONCLUSION: This in vivo system provides a new platform for studying human blood cell malignancies at much lower cost than other animal models and has the potential to provide rapid chemotherapy assays, which could significantly reduce drug development time and expense.