Adult-onset cyclic neutropenia is a benign neoplasm associated with clonal proliferation of large granular lymphocytes.

Human cyclic neutropenia occurs in children and adults. Adult-onset cyclic neutropenia is an acquired disease characterized by increased numbers of large granular lymphocytes (LGL), in contrast to childhood-onset cyclic neutropenia in which LGL counts are normal. We investigated the clonality of lymphocytes in these two groups of patients by assessing the rearrangement status of the T cell receptor beta chain gene. Patients with adult-onset cyclic neutropenia showed clonal rearrangement of the T beta gene whereas the children did not. Since LGL are known to have multiple regulatory effects on normal hematopoiesis, the finding of a clonal proliferation of this lymphocyte population implicates these cells in the pathogenesis of cyclic neutropenia.

Canine cyclic hematopoiesis is associated with abnormal purine and pyrimidine metabolism.

Canine cyclic hematopoiesis is an autosomal recessive disease characterized by regular 11-13-d cycles of the neutrophil, reticulocyte, and platelet counts caused by a defect in regulation of marrow stem cell proliferation. Treatment with lithium abrogates cycling of the cell counts in these grey collie dogs. Aware of the defective lymphopoiesis associated with adenosine deaminase and purine nucleoside phosphorylase deficiencies, we hypothesized that abnormal purine or pyrimidine metabolism might be present in these dogs. Using high pressure liquid chromatography, we measured erythrocyte purine and pyrimidine nucleotide levels and plasma purine and pyrimidine nucleosides and bases in normal and grey collie dogs before and during lithium treatment. During neutropenic periods in the grey collies, erythrocyte ATP, GTP, and UTP levels were significantly elevated. Normal dogs made neutropenic with cyclophosphamide did not show such elevations. Lithium treatment normalized the levels of erythrocyte ATP, GTP, and UTP in the grey collies and eliminated the differences between normal and grey collie nucleotide levels. Plasma thymine levels were markedly increased during neutropenia in the grey collie but were not increased in cyclophosphamide-treated normal dogs. The finding of abnormal concentrations of purine and pyrimidine metabolites in these dogs suggest that a metabolic derangement in purine or pyrimidine metabolism may be the cause of the defective stem cell proliferation in this disease.

Cyclic hematopoiesis. Effects of endotoxin on colony-forming cells and colony-stimulating activity in grey collie dogs.

Cyclic changes in blood neutrophil counts of grey collie dogs with cyclic hematopoiesis can be eliminated by daily endotoxin injections. Studies were performed to determine the mechanism whereby endotoxin alters this disease. Bone marrow granulocyte-macrophage progenitor cells (colony-forming cells [CFUc]) showed cyclic variation in the untreated grey collie, which was eliminated by chronic endotoxin treatment (Salmonella typhosa lipopolysaccharide W, 5 microgram/kg per day). Similar cyclic variation in blood CFUc was eliminated by this treatment. Tritiated thymidine suicide of the marrow colony-forming cells failed to show cyclic changes to explain the marked swing in CFUc numbers in untreated grey collies. The thymidine suicide rates were not significantly changed by chronic endotoxin treatment. Similarly, serum colony-stimulating activity did not show cyclic variation with the cyclic neutrophil counts in untreated grey collies and was not altered by chronic endotoxin treatment. We suggest that endotoxin eliminates neutrophil cycling in cyclic hematopoiesis by a direct effect on the flux of pluripotent stem cells into the committed stem cell compartment and that this occurs independent of changes in serum colony-stimulating activity.

Chronic neutropenia. A new canine model induced by human granulocyte colony-stimulating factor.

Normal dogs were treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) at 10 micrograms/kg/day for 30 d, which caused an initial neutrophilia, followed by a prolonged period of chronic neutropenia. A control dog treated with recombinant canine G-CSF (rcG-CSF) showed persistent neutrophilia over 3 mo. Serum from dogs during neutropenia contained an antibody to rhG-CSF, which neutralized the stimulatory effects of both rhG-CSF and rcG-CSF on dog marrow neutrophilic progenitor cell growth and on NFS-60 cell proliferation. 4 mo after discontinuation of rhG-CSF, the dogs' neutrophil counts returned to the normal range. Rechallenge with the rhG-CSF re-induced severe neutropenia in 1 wk. Neutropenia was transferred by plasma infusion from a neutropenic dog to a previously normal dog. These data suggest that human rhG-CSF immunizes normal dogs and thereby induces neutralization of endogenous canine G-CSF and neutropenia. This model system should allow more precise definition of the in vivo role of G-CSF.

Abnormal GM-CSA generation by lymphocytes in patients with cyclic hematopoiesis.

Human cyclic hematopoiesis (CH) is a disease characterized by regular 21-day cyclic fluctuations of blood cell counts due to fluctuations in bone marrow cell production. The regular periodicity of the fluctuations suggests a defect in a regulatory feedback control loop. We examined the production of monocyte-derived recruiting activity (MRA) by monocytes and the response to MRA of lymphocytes from three patients with CH. MRA production was normal or increased in patients' monocytes, but granulocyte-macrophage colony-stimulating activity (GM-CSA) production in response to MRA was decreased in lymphocytes from patients with CH (p = 0.005). These data suggest that the regulatory defect in CH may involve defective lymphocyte generation of GM-CSA, resulting in deficient production of mature neutrophils.

Cyclic hematopoiesis in dogs: studies of erythroid burst-forming cells confirm an early stem cell defect.

Studies of early erythropoiesis in dogs have been hampered by the lack of an efficient assay for canine erythroid burst-forming cells (BFU-E). We have developed a methyl-cellulose culture system for hematopoietic progenitors in dog marrow with a plating efficiency for BFU-E of 98 +/- 26 (SD) per 10(5) marrow mononuclear cells. We then applied this assay to the study of cyclic hematopoiesis in grey collie dogs, and regular fluctuations of colonies derived from erythroid colony-forming cells (CFU-E), BFU-E, and granulocyte-macrophage colony-forming cells (CFU-GM) were seen at 12- to 13-day intervals. Defining Cycle Day 1 as the first day that the granulocyte count falls below 1000/mm3, we found that the peak frequency of BFU-E (Cycle Day 10) always preceded the peak frequency of CFU-E (Cycle Day 12), which preceded that of the reticulocyte count (Cycle Day 3). The peak frequency of CFU-GM (Cycle Days 9-2) and neutrophil-containing colonies in agar culture (Cycle Days 1-2) preceded the peak of granulocytes (Cycle Day 6). The percentage of the various progenitors in the DNA synthetic phase of the cell cycle was similar in grey collies and normal dogs and showed no cyclic fluctuations. These data indicate that cyclic hematopoiesis results from a defect in a hematopoietic stem cell more primitive than BFU-E and CFU-GM. Cycling appears to be due to the commitment of this primitive cell to differentiation only at discrete intervals. The cell cycle kinetics and differentiation of subsequent cells appear normal.

Suppression of in vitro granulocytopoiesis by captopril and penicillamine.

The mechanisms underlying drug-induced neutropenia are poorly characterized. We have examined the mechanism of suppression of granulocytopoiesis by captopril and penicillamine using human and canine bone marrow cells in an in vitro culture system. Addition of captopril caused no significant change in granulocyte-macrophage colony formation at concentrations up to 30 micrograms/ml. In the presence of CuSO4 (1-3 micrograms/ml), however, captopril caused significant inhibition of colony growth (p less than 0.05). Penicillamine, another agent associated with neutropenia and, like captopril, having a reactive thiol group, also inhibited colony formation in the presence of copper. Chemical congeners of captopril lacking a reactive thiol group and enalaprilic acid, an alternative angiotensin-converting enzyme (ACE) inhibitor, failed to show inhibition, suggesting that the thiol group and not ACE inhibition was responsible. Analysis of day-7 colonies (98% neutrophilic) and day-21 colonies (37% neutrophilic, 30% macrophagic, 27% eosinophilic, and 6% mixed) showed that neutrophil-containing colonies, but not nonneutrophilic colonies were inhibited by the addition of captopril plus copper. Catalase totally reversed the inhibition of colony formation caused by these agents. Direct measurement of oxygen consumption in the presence of captopril showed marked enhancement with the addition of CuSO4 and a 48% reduction in the presence of added catalase. These data indicate that drugs with a reactive thiol group can interact with copper to generate H2O2, which can be toxic to neutrophilic progenitor cells. We postulate that this may be an important mechanism for drug-associated neutropenia and a general mechanism for drug-induced marrow cell injury.

Cyclic neutropenia: a clinical review.

Cyclic neutropenia is a benign, hematologic disorder characterized by recurrent episodes of severe neutropenia at 21 day intervals. There are associated cyclical variations in other blood cells. Patients with this disease have malaise, stomatitis, cervical lymphadenopathy and fever during the recurrent neutropenic periods. The exact cause of cyclic neutropenia is unknown. About one third of human cases appear to be inherited in an autosomal dominant pattern. In the other cases, the disease appears to arise spontaneously with symptoms usually beginning in infancy or early childhood. In adult patients, the disease may be acquired and occur in association with a clonal proliferation of large granular lymphocytes. Clinical studies in man and investigations in grey collie dogs, which have a very similar disease, strongly suggest that cyclic neutropenia is due to an abnormality in the regulation of early hematopoietic precursor cells. Therapy for cyclic neutropenia involves local and symptomatic therapy for the recurrent mouth ulcers and pharyngitis, and antibiotics for episodes of sinusitis, pneumonia, peritonitis, or bacteremia. Therapy with glucocorticosteroids, androgens, and plasmapheresis has been efficacious in a few adult patients, but no therapy has been proven to alter the cycling of blood counts in children. Despite their repetitive illnesses, patients with cyclic neutropenia grow and develop normally. With the help of attentive physicians and dentists, their quality of life and life expectancy are good. Current research on hematopoietic growth factors offers promise of new approaches to therapy.

Continuing medical education for life: eight principles.

Continuing medical education (CME) is being pressured to change in response to increasing and changing educational needs of practicing physicians, fostered by technical innovations, evolution of practice styles, and the reorganization of health care delivery. Leadership in the reform of CME falls primarily to the medical specialty societies in light of their traditional responsibilities for accrediting CME and maintaining professional standards. To address the need for reform, the American College of Obstetricians and Gynecologists in 1997 organized a conference to assemble CME program administrators from several medical specialties and academicians with expertise in postgraduate learning. At the conference, issues facing CME were examined. The authors, who were conference participants, state and explain eight principles that emerged from conference discussions. (For example: "Educational activities should be supportive of and coordinated with the transition to evidence-based medicine.") The principles reflect the interspecialty and interdisciplinary consensus achieved by the conference participants and can serve as useful guideposts for educators as they work to improve CME in their institutions. The authors conclude by noting the need for a more systematic and rigorously analytic approach, where CME content is determined according to assessed needs and CME is evaluated by measuring outcomes; for this to happen, CME educators and faculty must be brought up to date through training, including the use of problem-based learning. CME must also instill collegiality, interaction, and collaboration into the learning environment instead of being a solitary learning activity. Finally, CME must not only emphasize the acquisition of knowledge but also instruct physicians in the process of decision making to help them better use their knowledge as they make clinical judgments.

Osmotic stress in red blood cells from beagles with hemolytic anemia.

Red blood cell populations separated by density centrifugation were compared in a dynamic assay of osmotic stress. Red blood cells from Beagles genotypically normal and nonanemic (nonaffected), Beagles with inherited hemolytic anemia (anemic), and Beagles presumed to be carriers of the anemia trait (trait carriers) were examined for rate and extent of swelling after exposure to the ionophore A23187 in a medium containing calcium and potassium chloride. Comparisons were made between RBC populations separated on the basis of density. Significant differences were observed in the rates of cell swelling in RBC populations separated by density between nonaffected and anemic Beagles. The response of RBC from Beagles presumed to carry the anemia trait was similar to that of RBC from nonaffected dogs. One phenotypic expression of this inherited abnormality of RBC in Beagles was an accelerated rate of RBC swelling under osmotic stress, and this swelling response diminished with increasing RBC density.

Cyclic hematopoiesis: effects of lithium on colony-forming cells and colony-stimulating activity in grey collie dogs.

The cycling of blood cell counts in grey collie dogs with cyclic hematopoiesis can be eliminated by treatment with oral lithium carbonate. To explore the mechanism by which lithium alters this stem cell disorder, studies of bone marrow granulocyte-macrophage progenitor cells (CFU-C), neutrophil colony-forming cells (neutrophilic CFU-C), and colony-stimulating activity (CSA) were performed. In untreated dogs, the proportions of CFU-C were found to fluctuate cyclically, but the cyclic fluctuations in neutrophil colony-forming cells were even more marked, with numbers decreasing to undetectable levels during each period of neutrophilia. Dogs on lithium, however, did not cycle the numbers of total or neutrophilic CFU-C. Tritiated thymidine suicide rates were not altered by treatment with lithium. Serum CSA levels and bone marrow cell elaboration of CSA were not increased by lithium. These studies suggest that lithium corrects cyclic neutropenia by a direct effect on the differentiation and proliferation of CFU-C; normalization of the proportion of CFU-C that enter neutrophilopoiesis appears to be an important effect of the lithium therapy.

Lithium therapy of canine cyclic hematopoiesis.

Treatment of cyclic hematopoiesis in the grey collie dog with lithium carbonate eliminated the recurrent neutropenia and normalized the other blood cell counts. These findings suggest that human cyclic hematopoiesis may be successfully treated with lithium. The effects of lithium on the monocytes, platelets, and reticulocytes, as well as the neutrophils, suggest that lithium operates on basic regulatory mechanisms affecting the most primitive hematopoietic precursor cells.

Mechanism of canine cyclic hematopoiesis: the role of prostaglandin E in feedback regulation.

Prostaglandin E inhibits granulocyte-macrophage colony formation in vitro in man and mouse, suggesting that it plays a role in feedback regulation of granulocyte production in vivo. Therefore, we examined the role of PGE in normal canine hematopoiesis and its potential role in the pathogenesis of cyclic hematopoiesis in grey collie dogs. The prostaglandin synthesis inhibitors indomethacin and ibuprofen (10(-5) M) increased CFU-C growth to 194 and 160% of control, respectively, while PGE2 addition caused a dose-dependent inhibition of bone marrow CFU-C growth in both normal and grey collie dogs. These concentrations of indomethacin and ibuprofen decreased bone marrow cell elaboration of PGE measured by radioimmunoassay to less than 5% of control values. The levels of PGE in leukocyte conditioned medium prepared from grey collies correlated with the number of monocytes in the conditioning cell suspension (r = 0.78, n = 10, p less than 0.05) so that PGE production per monocyte was no different in normal and grey collie dogs. The effect of PGE2 upon CFU-C was to inhibit formation of macrophage, but not neutrophil colony subtypes. These findings make aberrant PGE-mediated inhibition of precursor cells an unlikely mechanism to cause cyclic hematopoiesis, and show that PGE produced by monocytes acts as a feedback inhibitor for precursor cells destined to produce monocytes but not for those destined to form neutrophils.

Human cyclic hematopoiesis is associated with aberrant purine metabolism.

It appeared possible that abnormal purine or pyrimidine metabolism could cause cyclic hematopoiesis by analogy with the defective lymphopoiesis associated with inherited deficiencies of adenosine deaminase and purine nucleoside phosphorylase. Therefore, we examined erythrocyte purine and pyrimidine nucleotide levels, as well as plasma purine and pyrimidine nucleosides and bases in three patients and in normal controls. These studies showed that during neutropenia there was a significant elevation in the levels of guanosine triphosphate (P = 0.005) and adenosine triphosphate (P less than 0.001) in the patients' red cells not attributable to reticulocyte variation. Serial analysis of a patient's plasma showed a fivefold elevation of hypoxanthine (10.6 mumol/L) during neutropenia, with a return to normal values (1.4 mumol/L) as neutrophil numbers increased. Plasma inosine was also significantly elevated in comparison with normal control values (2.0 mumol/L vs. 0.8 mumol/L), whereas plasma and urinary uric acid were within the normal range. Serial analysis of red cells and plasma from two patients with chronic neutropenia showed no elevations of purine or pyrimidine metabolites. These results provide evidence of a link between abnormal concentrations of purine metabolites and cyclic hematopoiesis, and permit the speculation that aberrant purine metabolism is primarily related to the defective hematopoietic cell proliferation that is characteristic of this disease.

Severe neutropenia in infectious mononucleosis.

Mild neutropenia is a well-known concomitant of infectious mononucleosis caused by the Epstein-Barr virus (EBV) occurring in the first weeks of illness. However, severe neutropenia (less than 200 polymorphonuclear leukocytes per mul) is not generally regarded as a complication of infectious mononucleosis. Three patients were seen with severe neutropenia and EBV infection, and an additional eight cases were found in the literature. In two of the latter cases the neutropenia was fatal. In the 11 cases the severe neutropenia began 14 to 40 days after illness and usually lasted for three to seven days. At the time of severe neutropenia, studies of marrow specimens showed increased proportions of promyelocytes and myelocytes. Our data suggest that EBV infection is the proximate cause of the severe neutropenia in some patients with infectious mononucleosis and that in such cases close observation and early treatment of suspected superinfections is necessary.

Canine cyclic hematopoiesis: effects of chronic endotoxin administration.

Endotoxin was given to grey collie dogs to investigate the mechanism of cyclic hematopoiesis. Over prolonged periods of daily administration, low doses of endotoxin (0.1 microgram/kg/day) failed to change the cycling of blood neutrophils or reticulocytes. However, higher doses of endotoxin maintained at a plateau level (5 microgram/kg/day) eliminated the cyclic changes in neutrophils and stabilized the reticulocyte and platelet counts in the normal range. Administration of endotoxin at this dose also eliminated the cyclic marrow differential count fluctuations characteristic of this disease. We infer from these results that endotoxin ameliorates the cyclical changes in blood cell counts by regulating hematopoietic proliferative activity at the stem cell level.

T(8;21) with a phenotype of chronic myeloid leukemia.

A myeloproliferative disorder having the phenotype of chronic myeloid leukemia (CML) with t(8;21) is reported. Neither t(9;22) nor a DNA rearrangement in the bcr region was detected. The presence of t(8;21), in contrast with previous experience, does not appear to prevent full maturation of the granulocytic series.

Canine cyclic haematopoiesis: the effect of endotoxin on erythropoiesis.

We have examined the effects of chronic endotoxin treatment on erythropoiesis in six grey collies with cyclic haematopoiesis. Blood reticulocytes, bone marrow erythroid colony (EC) forming cells, serum iron and erythropoietin (ESF) values showed regular periodic fluctuations in untreated grey collies. Daily endotoxin injections eliminated the cyclic fluctuations of reticulocytes and EC. The mean serum iron values were increased and recurrent hypoferraemia eliminated, while the mean serum ESF values were reduced. The cyclic fluctuations of serum ESF values were no longer apparent in the endotoxin treated grey collies. Tritiated thymidine suicide of the marrow EC forming cells failed to show cyclic changes either in untreated or endotoxin treated dogs. The ESF sensitivity of EC in the grey collie was unchanged during endotoxin treatment and was not different from normal dogs. Endotoxin appears to alter periodic erythropoiesis by stabilizing the flux of cells into the committed erythroid precursor cell pool from a more primitive stem cell compartment.

Lithium augments GM-CSA generation in canine cyclic hematopoiesis.

Cyclic hematopoiesis in gray collie dogs can be cured by lithium treatment. We examined the mechanism of lithium's effect by developing an assay for the canine equivalent of GM-CSF (called GM-CSA). Phytohemagglutinin (PHA)-stimulated canine blood mononuclear cells produce GM-CSA in a dose-dependent manner; this GM-CSA stimulates more neutrophil-containing colonies than does endotoxin-treated dog serum. Production of GM-CSA by PHA-stimulated normal dog cells was not altered by lithium. However, cells from gray collies during their neutrophilic period increased their GM-CSA when lithium (2 mEq/L) was added to low doses of PHA, whereas neutropenic gray collie cells did not. These data suggest that lithium could modulate cyclic hematopoiesis by increasing intramedullary GM-CSA at the time when marrow neutrophilic progenitor cells are at their nadir.