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[Figure][1] A 41-year-old man presented to our institution with a 2-week history of fever, diarrhea, and anorexia after a trip to Bangladesh. Physical examination revealed no jaundice, lymphadenopathy, or organomegaly. His blood cell counts were within normal range except for eosinopenia (0

Abstract 1237 Eosinophilia is a common clinical problem associated with numerous disorders such as atopic diseases, parasitic infections, hypereosinophilic syndrome (HES), and cancer. A marked decrease in circulating eosinophils, or eosinopenia, has long been associated with acute bacterial infections. Eosinopenia has been shown to be a sensitive and reliable marker for distinguishing between non-infectious and infection-associated sepsis in the intensive care unit setting. Eosinopenia with acute infection had been assumed to be secondary to the release of adrenal glucocorticoids in response to the stress of infection. However, animal studies performed in the 1970s demonstrated that inhibition of parasite-associated eosinophilia with acute bacterial infection was independent of adrenal stimulation; the investigator speculated that it was due to a direct effect on the eosinophil precursor. To date, the mechanism of infection-associated eosinopenia, and its potential for suppressing IL-5-mediated eosinophilia, has not been investigated. To study eosinophil development, we established an ex vivo liquid culture system in which we can follow the differentiation of murine eosinophil lineage-committed progenitors (EoPs) to mature functional effector eosinophils that express known eosinophil surface markers and granule proteins and respond to eosinophil-active chemoattractants. We demonstrate that murine eosinophil precursors express mRNA for six TLRs. Ligands for TLR2 heterodimers, TLR4 and TLR7, but not TLR3, inhibited eosinophil ex vivo growth by attenuating EoP proliferation in response to IL-5 stimulation. Exposure to heat-killed E. coli also resulted in diminished EoP proliferation. The developing eosinophils further responded to TLR activation with production of a subset of inflammatory cytokines, including IL-6, IL-10, TNF-alpha and CXCL10. Co-culturing of untreated EoPs with LPS-exposed EoPs resulted in reduced IL-5-mediated proliferation of the untreated EoPs, suggesting that EoPs secrete mediators in response to TLR activation that regulate their proliferation in an autocrine/paracrine manner. Neutralization of IFN-beta, but not IFN-alpha or IFN-gamma, partially protected developing eosinophils from the inhibitory effects of LPS. In vivo studies revealed that endotoxemia and bacteremia reduced numbers of EoPs in the bone marrow of wild-type mice. Further, LPS dose-dependently reduced hypereosinophilia in IL-5 transgenic mice, highlighting the potential therapeutic value of this approach even in an extreme IL-5-driven clinical state. Suppression of eosinophil production by TLR4 ligands is conserved between mice and humans as yield of eosinophils from human EoPs was reduced following LPS exposure. Taken together, these findings identify a mechanistic explanation for eosinopenia following bacterial infections and identify a novel therapeutic strategy for inhibiting eosinophil production and peripheral eosinophilia in eosinophil-associated diseases. Disclosures: No relevant conflicts of interest to declare.

Abstract 1558 Eosinophils and their progenitors are robustly elevated in the bone marrow in response to diverse stimuli including helminth infection and allergic triggers. In contrast, bacterial infection results in diminution in the number of circulating eosinophils. Markedly reduced peripheral blood eosinophils of sudden onset but prolonged duration is generally a consequence of acute bacterial infection. Further, eosinopenia has been shown to be a sensitive marker of sepsis and correlates with severity of disease in critically ill patients. While the initial eosinopenic response is believed to be secondary to rapid migration of circulating eosinophils to the site of infection, the mechanism for prolonged eosinophil depletion with bacterial infection remains undefined. As Toll-like receptors (TLRs) have been shown to be expressed by early hematopoietic progenitors and by mature eosinophils, we initially investigated TLR expression during eosinophil differentiation. In a culture system we developed to differentiate mature eosinophils from low density bone marrow progenitors, IL-5 stimulation of progenitors resulted in induced expression of seven TLRs, including TLR1 (4-fold), TLR4 (5-fold), TLR6 (2-fold), TLR7 (6-fold), TLR8 (4-fold), TLR9 (3-fold), and TLR13 (8-fold), when compared to expression in the progenitors prior to IL-5-induced differentiation. Notably, a majority of the TLRs (TLR1, TLR4, TLR7, TLR8, TLR9, and TLR13) were induced specifically early in eosinophil differentiation after 4 days of IL-5 stimulation and prior to expression of surface markers, including Siglec-F and CCR3, associated with mature eosinophils. To study the effects of TLR signal transduction on eosinophil development, we exposed bone marrow progenitors to TLR agonists for 18 hours after 4 days of IL-5 stimulation and then subsequently induced further eosinophil differentiation with IL-5 stimulation for another 8 days. We measured effects of TLR signaling on cell number, eosinophil differentiation, cytokine production and protease activity. LPS stimulation resulted in a reduction in total cell numbers by more than 65% with immature eosinophils a predominant cell type. In addition, LPS stimulation of progenitors resulted in significantly increased cytokine and chemokine production, including IL-6 (80-fold) and CCL22 (2-fold), and protease activity (2-fold) after 4–6 days of subsequent IL-5 stimulation compared to IL-5 stimulation alone. Similarly, stimulation of eosinophil progenitors with the TLR1/TLR2 agonist PAM3CSK4 or TLR6/TLR2 agonist PAM2CSK4 for 18 hours resulted in markedly decreased total cell numbers in a dose-dependent manner after 3 days of subsequent IL-5 stimulation. Together, these data suggest that TLR signaling in progenitors results in inhibition of IL-5-stimulated eosinophil development with aberrant cytokine, chemokine and protease production. As eosinophil granules have been shown to have anti-bacterial properties and eosinophilia improves survival rate in an experimental bacterial peritonitis model, delineation of the mechanism of infection-induced eosinopenia may lead to novel adjuvant therapeutics for bacteremic critically ill patients. Disclosures: No relevant conflicts of interest to declare.

Hypereosinophilic syndrome (HES) was a group of diseases associated with persistent eosinophilia without unknown causes, companied with tissue and organ impairments. In 2001, WHO classification of hematopoietic and lymphoid neoplasms classified chronic eosinophilic leukemia (CEL) / HES into the category of chronic myeloproliferative disease, and proposed that the principal basis for the identification of CEL and HES was whether to have the evidence of eosinophils clonal proliferation: CEL had the evidence of eosinophils clonal proliferation, while HES was lack of the evidence of eosinophils clonal proliferation. In this study, 20 cases of CEL/ HES patients were analyzed retrospectively, and nested RT-PCR was used to detect FIP1L1-PDGFRA (F/P) fusion gene; Allele-specific PCR (ASP) conjoint sequencing analysis was used to detect JAK2 V617F, and PCR-RFLP was adopted to detect the mutation status of JAK2 V617F; and PCR is applied to detect TCRγ rearrangement. The clinical and laboratory characteristics of CEL and HES were compared. The ratio of male and female in the 20 cases of patients was 19:1, and the median age was 33 (20–57). F/P detection was positive for 12 cases, and the sequencing confirmed that FIP1L1 break point was at intron 10–12, while PDGFRA break point was at exon 12. There was 1 case of patient found that had JAK2 V617F, and the mutation status analysis showed that it was the mutation on heterozygote. 6 cases were detected having TCRγ gene rearrangement, of which 4 cases were CEL patients, and other 2 cases were HES patients. Most of CEL patients had respiratory symptoms in the early stage, easily companied with circulatory systematic impairment and nervous systematic symptoms. The incidence of splenomegaly of CEL patients was obviously higher than that of HES patients (92.5% vs 42.5%, p=0.031), so did the incidence of anemia and myelofibrosis. There was no difference in EO, WBC, PLT and EO% in peripheral blood as well as bone-marrow eosinophils percentage and bone-marrow primitive cells’ percentage between two groups. Abnormal morphology of eosinophils was often found in CEL patients, with the main manifestation of eosinopenia, basopenia and plasma vacuoles. Our data showed that Eosinophilia is often caught by male, mainly by the young; CEL patients have the main manifestation of the circulatory systematic, respiratory and gastrointestinal symptoms, and have a high incidence of anemia and thrombocytopenia. The routine examination has a little significance for the identification o CEL and HES, while the bone marrow smears morphological examination has a certain help for the diagnosis of CEL; Some HES patients have JAK2 V617F mutation, and further studies on the effect of JAK2 V617F mutation on the pathogenesis of HES can contribute to the diagnosis of such patients in the future and the development of the new targeted drug therapy; Some CEL patients have TCRγ rearrangement, while the relationship of CEL and TCRγ needs a further study.

1905, itwas not until 1948 that Godlowski5 and Laragh and Almy7 reported the eosinopenia that occurs in normal subjects some two hours after epinephrine is injected. At the present time, the changes in the leukocytes resulting from the injection of epinephrine need to be re-examined in the light of the changes known to be produced by adrenocortical hormone and by adrenocorticotrophic hormone; for the work of Vogt,’3 of LongS and of Sayers and Sayers” indicates that epinephrine leads to an increased output of these two hormones. In the course of this re-examination we have obtained some new qualitative and quantitative information about the effect of epinephrine on the leukocyte count of human beings. The earlier literature on the subject has been summarized by Garrey and Bryan,3 and there is fairly wide agreement upon the general features of the reaction. The lymphocytes usually increase quickly, and then within one hour fall below the initial level; but if the dose of epinephrine is very large, the lymphocytosis persist for as long as six hours. There is also a neutrophilia which develops more slowly and lasts for a longer time than in the usual type of lymphocytosis. One of the most interesting additions to the literature since the above review appeared is the finding of the late eosinopenia already referred to. Other contributions come from Lucia, Leonard and Falconer,9 who have shown that patients from whom the spleen has been removed respond to epinephrine in the same way as do normal subjects, and from Gabrilove, Volterra, Jacobs and Soffer,4 who found that normal subjects and patients with Addison’s disease react in the same way, so far as their leukocytes are

mone on white blood elements in mice and other mammals is well known,’ and has been partially reviewed by Gordon.8 Lymphopenia, lysis of lymphoid tissue, eosinopenia, granulocytosis and myeloid metaplasia occur in various combinations, the particular configuration depending upon the animal Species. In mice, granulocytosis and myeloid mnetaplasia are particularly striking (luring hormone therapy.�7 This led us to test the effect of long-term, intermittent cortisone therapy on mice, in the hope that permanent and irreversible changes might occur. Our preliminary experiments indicated that the granulocytosis might become permanent and not dependent upon continuing cortisone therapy. The experiments reported here confirmed this finding. In addition, a few animals developed apparent myeloid leukemia in the spleen and liver.

A technic of performing reliable eosinophil determinations on the peritoneal fluid of mice is described. This procedure was found to be rapid and did not necessitate killing the animals. Simultaneous determinations of the blood and peritoneal eosinophils were made at 3 hour intervals throughout a 24 hour period. A marked diurnal rhythm was found in the blood eosinophils, but little change occurred in the number of eosinophils found in the peritoneal cavity. During a period of 3 hours following stress, there was a marked blood eosinopenia, but little or no change in the peritoneal eosinophils. Repeated intraperitoneal injections of 0.1 ml. normal horse serum resulted in a great increase in the number of peritoneal eosinophils. These cells appeared to be morphologically identical with the blood eosinophils. In mice which were pretreated with horse serum, relatively large injections of cortisone, compound F, epinephrine and histamine produced a marked blood eosinopenia, but failed to reduce the number of peritoneal eosinophils within 3 hours. The response of eosinophils in blood and peritoneal fluid to cortisone was essentially the same either when it was injected subcutaneously or intraperitoneally. Implantations of pellets of cortisone acetate in the peritoneal cavity produced a blood eosinopenia beginning within 1 hour and reaching a maximum at approximately 3 hours. The peritoneal eosinophils did not begin to drop until approximately 6 to 10 hours after the implantation. These data were interpreted as indicating that physiologic doses of cortisone, compound F, epinephrine or histamine do not have a direct lytic action upon the eosinophils of mice.

Hemolysis was induced in rats by the intravenous administration of phenylhydrazine, immune hemolytic serum and Tween 80. During the period of red cell destruction, which lasted for three to four days, there was a marked leukocytosis, neutrophilia and eosinophilia which also persisted for three to four days. With phenylhydrazine and Tween 80 there was an initial transitory eosinopenia; with immune serum there was an immediate eosinophilia. In contrast, anemia produced by cardiac hemorrhage was accompanied by a transient slight leukocytosis and eosinopenia. Within one day the white cell level returned to baseline levels, but eosinophilia appeared and persisted for two days. The injections of formaldehyde, ACTH, and saline, and cardiac puncture without bleeding caused a slight ephemeral leukocytosis and eosinopenia. The peripheral blood cell response observed during hemolysis occurred in the presence or absence of the adrenals, and with or without the administration of ACTH. It was concluded that hemolysis elicits a distinct pattern of cellular response in the peripheral blood which is not mediated through the adrenal cortex.

1. Detailed hematologic observations, bone marrow aspirations and blood volume determinations were made on 20 patients with rheumatoid arthritis and allied disorders before, during and after the administration of either ACTH or cortisone. 2. Significant reticulocytosis occurred in every patient during therapy, but its magnitude was poorly correlated with either the initial degree of anemia or subsequent increase in circulating red cell mass. 3. There was an increase in hematocrit and total circulating red cell mass of all anemic patients who responded clinically to either ACTH or cortisone. There was little or no improvement of anemia when the clinical response was poor. 4. Polycythemia did not occur in any patient during prolonged therapy or with repeated courses of either ACTH or cortisone. 5. Hemodilution and hemoconcentration were much more profound during and after ACTH administration than they were with cortisone. 6. Bone marrow studies revealed moderate depression of the erythroid series before treatment. At the end of therapy erythroid elements were normal. 7. Significant polymorphonuclear leukocytosis occurred its all patients during therapy while lymphopenia was inconstant and unsustained. Circulating eosinophils were depressed more with ACTH than with cortisone treatment. 8. Before treatment eosinophils and their precursors were present in the bone marrow its normal or increased numbers. During therapy the number of these cells was unchanged in the marrow, even when there was profound peripheral eosinopenia 9. The role of ACTH and cortisone in the physiologic mechanism of hematopoiesis is discussed. 10. The improvement in the anemia associated with inflammatory disease in response to ACTH or cortisone therapy probably is a reflection of the control of the underlying disease rather than a primary "stimulation" of the bone marrow.

A study is reported of the influences exerted on the levels of the circulating eosinophils, lymphocytes and neutrophils of rats in which body temperatures have been elevated for one hour to 41.0 or to 41.5 C. by means of the Raytheon microwave diathermy apparatus. Preliminary studies were made to determine the influence exerted on these blood categories by the restrictions imposed on the animals such as confinement in cages and insertion of rectal thermistors: factors incident to the experimental procedure. Data were assembled on intact animals, trained to cage confinement and to thermistor implacement, and on such animals subjected to the elevated temperatures. Data were also assembled on adrenalectomized animals, hypophysectomized animals, hypophysectomized-adrenalectomized animals and adrenal-demedullated animals. These data warrant the following conclusions: 1. Cage restrictions and the insertion of the thermistor into the rectum resulted in a mild form of stress inducing a slight eosinopenia, a lymphopenia and a mild neutrophilia. The energy of the microwaves, applied for one hour, greatly accentuated those changes, resulting in marked eosinopenia and lymphopenia, with a greatly increased neutrophilia. When temperatures ranging from 41.0 to 41.5 C. were induced for one hour in previously trained rats, the total eosinophil tabulation decreased in four hours from a control level of 171 ± 24 cells per cu. mm. of blood to 41 ± 5 cells, a decline of 76 per cent. The total number of mononuclear leukocytes decreased from a control level of 19,940 ± 1,000 cells to 8,330 ± 810 cells immediately after heating, while the total number of neutrophilic leukocytes increased from a control level of 3,580 ± 650 cells per cu. mm. to 8,770 ± 1,920 cells at two hours after heating. 2. Adrenalectomy altered the hemograms of rats subjected to those elevated temperatures, by abolishing the eosinopenic and the lymphopenic trends but it did not alter the neutrophilia. 3. Removal of the pituitary gland from animals prior to the increases in their temperatures resulted in an average decrease in the number of eosinophils, a decrease which was not considered significant. The number of neutrophilic leukocytes remained high. 4. When animals lacking both pituitary gland amid adrenal glands were subjected to the same energy for one hour no significant alterations in the number of eosinophils, of lymphocytes or of neutrophils were recorded. 5. The adrenal medulla did not appear to play a significant role in the chain of events leading to the characteristic hemogram in animals subjected to fever. In adrenal-demedullated animals an eosinopenia, a lymphopenia and a neutrophilia occurred as in normal intact animals. 6. The conclusion seems warranted that hyperpyrexia has produced pituitary and adrenocortical stimulation.

1. Arteriovenous eosinophil differences across the spleen were measured in 10 dogs during the development of adrenal-mediated eosinopenia. In 7 instances the stimulus was epinephrine, in 2 ACTH, and in 1 operation alone. 2. In no case was significant net splenic uptake of eosinophils indicated. In 3 out of 7 epinephrine experiments and in 1 ACTH experiment, a significant net splenic output of eosinophils was found. For all 10 experiments averaged, the splenic output of eosinophils was significant. 3. Arteriovenous neutrophil and hematocrit differences were insignificant and were independent of simultaneous eosinophil differences. 4. It is concluded that the spleen is not a significant site of uptake on destruction of eosinophils during the development of adrenal-mediated eosinopenia. Under certain conditions, it actually extrudes eosinophils into the general circulation at a time when other factors are causing eosinophil disappearance. 5. The results suggest but do not establish the presence of a discriminatory splenic mechanism for the control of blood cells, capable of storing cells of one type while it releases others into the circulation.