Crizanlizumab in vaso-occlusive crisis caused by sickle cell disease.

Sickle cell disease (SCD) is a genetic disorder characterized by a single gene mutation leading to polymerization of erythrocytes, with subsequent assumption of a "sickle" shape. However, polymerization is just one aspect of the disorder's pathology. It is also characterized by abnormalities in nitric oxide utilization and vasculopathy; generation of reactive oxygen species; hemolysis; hypercoagulability; and altered rheology including abnormal leukocyte rolling along endothelium, and sickle cell-endothelial and cell-cell adhesion. The latter phenomenon is associated with increased P- and E-selectin expression and creation of a proadhesive endothelial environment. The anti-P-selectin humanized monoclonal antibody crizanlizumab functions through selective inhibition of P-selectin. At a dose of 5 mg/kg in a clinical trial, it led to a 45.3% decline in the median annual crisis rate in individuals with SCD. Tolerability was good and the adverse event profile was acceptable. In this review, results of the clinical trials involving this drug and specific side effects are outlined. Analysis of cost efficacy is touched on, with an examination of the economic and social burden of SCD.

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on primitive hematopoietic stem cell (PHSC) function and numbers, after chemotherapy.

OBJECTIVE: Primitive hematopoietic stem cell function was assessed after cyclophosphamide with granulocyte-macrophage colony-stimulating factor (GM-CSF), with or without preadministration of interleukin-1, using competitive repopulation. METHODS: C57B6/J mice injected with one or four biweekly intravenous injections of cyclophosphamide, 200 mg/kg, received granulocyte-macrophage colony-stimulating factor, 1 microg, subcutaneously for 5 days, beginning 24 hours after cyclophosphamide. Alternatively, mice were injected with interleukin-1, 1 microg, 20 hours before administration of drug or drug and cytokine. Marrow obtained from mice sacrificed 4 weeks after the last dose of drug or drug and cytokine was used in competitive repopulation. RESULTS: Significant reductions in marrow repopulating ability occurred after a single dose of cyclophosphamide or multiple injections. Repopulating units (RU) were calculated, and both binomial and Poisson models for estimation of primitive hematopoietic stem cell (PHSC) numbers were used. RU were significantly diminished for all treatment groups when compared to controls. PHSC numbers were not significantly affected by either regimen of cyclophosphamide given alone. Addition of GM-CSF to cyclophosphamide, whether the latter was given in single or multiple doses, led to further, although insignificant, declines in repopulating ability, as well as PHSC and RU numbers. Interleukin-1 usage exacerbated the observed repopulating defect. There was evidence of replicative failure in individual cells, indicating a qualitative defect also. SUMMARY: Additive stem cell depletion and qualitative replicative defect occur after chemotherapy-cytokine usage. However, the replicative defect of PHSC seen after addition of GM-CSF is not significantly worse than that seen with cytotoxic drug use alone.

Analysis of the stem cell sparing properties of cyclophosphamide.

OBJECTIVES: Cyclophosphamide was examined for its ability to spare the most primitive hematopoietic stem cell (PHSC). METHODS: C57BL6/J mice (Groups A and B) were sacrificed 24 h and 4-6 wk, respectively, after a single or second injection of low-dose cyclophosphamide (90 mg/kg) on days 1, 3, 7, or 15. A competitive repopulation assay was then performed, using B6-HbbdGpi-1a competitor cells, to determine the repopulating ability of exposed PHSC. RESULTS AND CONCLUSIONS: PHSC function was preserved after a single injection of cyclophosphamide and after a second injection on days 7 and 15 in both groups. In Group A, PHSC repopulating ability declined after a second injection on days 1 and 3 (p<0.05 only for day 1), as did repopulating units [RU]; PHSC numbers did not. In Group B, an insignificant decrease in repopulating ability and RU numbers was observed after a second injection on days 1 and 3, suggesting different etiologies for losses in the 2 groups, or correction of drug-induced defects within 1 month of cyclophosphamide administration. Total RU increased in single, day 1, 7 and 15 treatment groups. A significant number of marrow cells entered the S phase after cyclophosphamide dosing on day 3, and it is possible that a relationship exists between cell cycling and replicative damage. DNA damage was also increased 1 and 3 d after cyclophosphamide administration, although the significance of differences from controls was not definitive. CONCLUSION: Low-dose cyclophosphamide can spare stem cells, depending upon the timing of subsequent doses.

Long term hematopoietic damage after chemotherapy and cytokine.

Cancer chemotherapy causes severe damage to hematopoietic stem cells in both experimental animals and humans. While all levels of differentiation may be impacted, the most pivotal target of damage is the most primitive hematopoietic stem cell, PHSC. This cell not only suffers defective repopulating activity but also is quantitatively depleted. The causes of this damage are not clear. Severe possible explanations for this damage are discussed. They include: ineffective stromal support of stem cell function and reproduction; residual DNA damage preventing replication; accelerated cycling; and decreased responsiveness to normal physiologic growth stimuli. Efforts at chemoprotection, including manipulation of glutathione or aldehyde dehydrogenase levels, cytostatic peptides, immunomodulatory chemicals and cytokines are detailed. In particular, concern has been raised regarding potential deleterious consequences of combined chemotherapy-cytokine use, but substantiation of the cited data is warranted.

Interferon-gamma (IFN-gamma) as a potential radio- and chemo-protectant.

Interferons (IFN) have had increasing clinical usage in the treatment of a variety of disorders, at times being used in combination with chemo- or radiotherapy. However, interferons may have inhibitory effects on hematopoietic stem cell proliferation and the effects of this cytokine's use on long-term hematologic function have not been studied. We performed the competitive repopulation assay in the murine system, using cells exposed to irradiation or single-dose chemotherapy with or without concomitant IFN-gamma use. IFN-gamma alone had no deleterious effects on hematopoietic stem cell productivity. We measured the repopulating ability of exhaustible multilineage precursors that were present at early stages of marrow repopulation after competitive repopulation (30 days). These progenitors were minimally impacted by cyclophosphamide (CTX) with or without IFN-gamma. Irradiation (XRT) and CTX alone produced significant repopulating defects in the most primitive hematopoietic stem cell, PHSC. Addition of IFN to either treatment regimen resulted in protection of PHSC, with improved repopulating ability, although the levels of donor marrow reached control levels only when CTX and IFN were used together. The results of multiple use of IFN with chemotherapy must be studied further, but IFN may offer hematologic radio- and chemoprotection, in addition to its antitumor properties in clinical protocols for treatment of cancers.

Stem cell factor improves the repopulating ability of primitive hematopoietic stem cells after sublethal irradiation (and, to a lesser extent) after bone marrow transplantation in mice.

Bone marrow transplantation (BMT) and sublethal irradiation (XRT) cause profound long-term damage to hematopoietic stem cells. We used the competitive repopulation assay in mice to test the ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF), cytokines given in clinical settings to enhance marrow recovery after XRT or BMT and to protect the marrow repopulating ability of primitive hematopoietic stem cells (PHSC) after these modalities. The repopulating ability of exhaustible multilineage progenitors (EMP) was also tested after these modalities, with or without cytokines. Repopulating abilities of EMP and PHSC were significantly reduced after XRT or BMT; PHSC were preferentially affected. Administration of SCF to C57B6/J mice after XRT resulted in improved EMP and PHSC repopulating ability, although progenitor numbers--repopulating units--were not completely returned to control levels. Whether given as a single dose or multiple doses, GM after XRT did protect PHSC function from the deleterious effects of XRT, but this was not a significant effect. SCF caused an increase in PHSC repopulating ability after BMT, but this too was not a significant difference. GM after BMT had little effect. SCF administration before XRT led to severe impairment of PHSC function with very little or no stem cell activity observed. Therefore, timing of its administration is an important consideration since preadministration of the cytokine before XRT can be extremely harmful to PHSC function.

Hematopoietic precursor cell exhaustion is a cause of proliferative defect in primitive hematopoietic stem cells (PHSC) after chemotherapy.

The authors used the competitive repopulation assay and simple statistical analyses to estimate concentrations of primitive hematopoietic stem cells (PHSCs) in the marrow of mice after chemotherapy. Single doses of cyclophosphamide (CTX) from 80 to 200 mg/kg were administered to C57B16/J mice. Other treatment groups included mice given multiple doses of CTX at the lowest dose of 80 mg/kg; mice given four weekly doses of vincristine (VCR) or vinblastine (VBL); mice given two biweekly doses of bleomycin; mice receiving cytosine arabinoside (ARA) administered intraperitoneally thrice daily or as a continuous infusion by Alzet pump for 3 days; and controls given no drug. The lowest dose of CTX (80 mg/kg), given once or repeatedly, spared PHSC numbers and function. The functional capacity of PHSCs declined significantly once doses of CTX exceeded 100 mg/kg. Decreases in PHSC function were usually associated with reductions in PHSC numbers; repopulating units, which include all repopulating cells, were similarly reduced. At the highest dose (33 mg/kg for 3 days), ARA caused a decline in marrow repopulating function. Drugs associated with mild clinical myelosuppression, such as VCR and VBI, did not significantly affect the repopulating ability of PHSCs, although VCR caused drastic declines in PHSC numbers. The marrow reconstitutive defects clinically-observed after chemotherapy may be caused partly by depletion of the PHSC pool.

Renal collecting duct carcinoma in an 8-year-old child.

A fatal collecting duct carcinoma, presenting with pleural metastases, arose from the right kidney in an 8-year-old child. A distal nephron origin of the tumor is supported by positive tumor staining with Ulex europaeus and Arachis hypogaea, and a lack of staining with Tetragonolobus lotus. The ultrastructural features of short stubby microvilli, smooth basal cell membranes, and lateral membrane infoldings also support a distal nephron origin (inner most inner medullary collecting duct). This rare childhood renal neoplasm behaved similarly to that reported in adults with metastatic disease at presentation and a short fatal clinical course.

The identification of the single-stranded DNA-binding domain of the Escherichia coli RecA protein.

To identify the ssDNA-binding domain of Escherichia coli RecA protein, we examined the ssDNA-binding capabilities of synthetic peptides, the sequences of which were derived from the C- and N-termini and from sequences within loops L1 and L2 of the RecA molecule identified from the crystal structure. Synthetic peptides derived from amino acid residues 185-219 of several bacterial RecA proteins, which include loop L2 of RecA, bound to ssDNA in filter-binding assays, whereas three separate synthetic peptides corresponding to single point mutants of E. coli RecA in this region did not. The binding of RecA to ssDNA examined using a gel-shift assay was inhibited by a synthetic peptide derived from this ssDNA-binding region, but not by synthetic peptides derived from amino acid residues 301-329 of the C-terminus or from N-terminal residues 6-39. A peptide corresponding to amino acid positions 152-169 of the RecA molecule and spanning loop L1 and its flanking regions did not bind ssDNA at peptide concentrations up to 250 microM. We have also defined a synthetic 20-amino-acid peptide that comprises amino acid residues 193-212 and includes loop L2 of RecA as the minimum unit that can bind to ssDNA from this region of RecA. Finally, two maltose-binding protein-RecA fusion proteins were made, one containing amino acid residues 185-224 of RecA and the other the last 51 C-terminal residues of RecA (amino acid residues 303-353). In contrast to the C-terminus-derived fusion protein, the fusion protein containing the putative DNA-binding site demonstrated significant binding to single-stranded oligonucleotides in both filter-binding and gel-shift assays. These findings suggest that a portion of the region extending from amino acid residues 193-212 is either part of or the whole ssDNA-binding domain of the RecA protein.

Assessing permanent damage to primitive hematopoietic stem cells after chemotherapy using the competitive repopulation assay.

The competitive repopulation assay was used to document the effects of six chemotherapeutic agents on primitive hematopoietic stem cells. The assay measures the relative abilities of donor cells to produce circulating erythrocytes and lymphocytes in lethally irradiated congeneic mice over a period of 6 months. Long-lasting marrow reconstitutive deficits in cells of donor origin occurred after exposure to 5-fluorouracil (5FU), bis-chloronitrosourea (BCNU), cyclophosphamide (CTX), vincristine (VCR), and actinomycin D (ACT) but not after exposure to cytosine arabinoside (ARA). Repopulating abilities were reduced after as little as a single dose of CTX or BCNU. A second dose of BCNU caused even more severe effects. A single dose of 5FU had no effect on repopulating abilities despite a temporary 10-fold reduction in marrow cell number, but multiple doses reduced the marrow stem-cell replicative ability to less than half of the normal control levels. These effects were not reliably predicted or detected by colony-forming assays or by reductions in marrow cell number. Thus, long-lasting proliferative defects in the primitive hematopoietic stem-cell (PHSC) population can result from the use of chemotherapeutic agents. Such findings may have clinical implications, especially in individuals receiving repeated or prolonged administration of these agents or in instances of marrow transplantation.

The University of Florida sickle cell screening program for neonates: design and results.

During the first 18 months of a pilot program for sickle cell screening at the University of Florida College of Medicine, Gainesville, 2,058 black neonates were screened. An incidence of homozygous sickle disease of 0.5 percent was greater than that expected or predicted by carrier frequency (8.3 percent). Fifty percent of all infants with abnormal cord blood electrophoreses were retested. All infants with actual homozygous disease or other clinically significant variants had confirmation of their diagnosis and were channeled for appropriate care. A change of phenotypic diagnosis based on a follow-up sample was made in eight cases. Errors were either interpretational or through contamination of cord blood samples by maternal blood at the time of delivery. Although location of infants for retesting after discharge was made more difficult by the largely rural composition of the target population, certain measures were taken to improve patient retrieval: use of public health personnel; enlistment of the aid of private physicians in the community; and inclusion of information regarding the screening program in the hospital discharge packets of black mothers. It is concluded that screening programs serving rural populations can adequately identify infants with abnormal hemoglobin patterns while educating and caring for families of these infants in a cost-efficient and effective manner.

The decrease in long-term marrow repopulating capacity seen after transplantation is not the result of irradiation-induced stromal injury.

Marrow cells from nonirradiated F1-W/Wv mice repopulated slightly less well than cells from lethally irradiated recipients. Therefore, avoiding irradiation of recipients did not improve the relative repopulating ability of their marrow cells. In other experiments, F1-W/Wv mice were transplanted by parabiosis with marrow of WBB6F1-+/+ (F1-+/+) mice, avoiding cellular handling and irradiation. Marrow cells transplanted to F1-W/Wv mice by this procedure demonstrated slightly better repopulating ability than did marrow cells transplanted by injection. However, they performed no better than those transplanted by parabiosis to irradiated F1-+/+ recipients. Significant impairment of stromal function after irradiation was not indicated. Apparently, stem cell damage caused by transplantation may have greater importance in causing loss of stem cell replicative potential than effects of irradiation-induced stromal injury.

The role of eosinophil in regulation of granulopoiesis.

An association between eosinophilia and neutropenia has been observed in a number of clinical conditions. To probe the role of eosinophils in granulopoiesis, marrow and peritoneal eosinophils, obtained from Schistosoma mansoni-infected mice, were separated and purified. Normal bone marrow cells were cultured in semi-solid culture medium in concentrations ranging from 5 X 10(4) to 5 X 10(5), with and without added eosinophils. To examine whether high prostaglandin E (PGE) content of eosinophils affects granulopoiesis, indomethacin was added to duplicate marrow cultures containing eosinophils. The addition of eosinophils to normal syngeneic marrow culture caused a significant inhibition of granulocyte-macrophage colony formation (CFU-GM) in culture. This suppressive effect was reversible upon addition of indomethacin. These findings suggest that eosinophils, in vitro, are capable of inhibition of granulopoiesis. The reversal of this effect by indomethacin indicates that this suppression may be prostaglandin mediated.

The effect of T cells from patients with infectious mononucleosis on CFU-CGM proliferation: a preliminary report.

The neutropenia occurring during infection is a poorly understood phenomenon. Immunologically-stimulated T lymphocytes, acting upon normal bone marrow stem cells, have been etiologically implicated in several disorders. Fifteen patients, ages 17 to 25 years, and diagnosed with infectious mononucleosis by positive heterophile titers, were studied. Peripheral blood T lymphocytes were separated using sheep red blood cell rosetting. They were then cocultured with normal bone marrow cells, in a concentration of 2 X 10(4) cells/ml, in methylcellulose containing 10% colony-stimulating activity. Normal BM was obtained from patients with nonmalignant hematologic disorders, or leukemia in remission. Bone marrow cells were cultured at a concentration of 1 X 10(5) or 5 X 10(5) cells/ml, alone (control) or with T lymphocytes. Plates were incubated at 37 degrees C with 5% CO2. Colonies were scored at 14 days. Inhibition of normal, bone marrow growth was observed at both concentrations, after addition of T lymphocytes to the culture system. Such suppression was significant (p less than 0.05) for the lower concentration of normal bone marrow cells only. Variable and partial abrogation of effect was seen after overnight incubation of T lymphocytes, possibly due to loss of suppressor activity. There were insufficient numbers of tests with supernatant to allow computation of statistical significance. Correlation between T-cell ratios and suppressive effect has not been determined, although it is suspected that the responsible cells are within the T-suppressor fraction.

Salmonella vertebral osteomyelitis and epidural abscess in a child with sickle cell anemia.

A case of Salmonella vertebral osteomyelitis with epidural abscess in a child with sickle cell anemia is presented. Spinal osteomyelitis is a rare event in children. Although osteomyelitis in sickle cell anemia may occur in any bone, it has most often been documented as beginning in the medullary cavity of the long and tubular bones. This is in contrast to the clinical presentation of osteomyelitis in the normal individual, who is likely to have infection beginning in and restricted to the metaphyseal regions of bones. Nonspecific or constitutional symptomatology may obscure the diagnosis of vertebral infection with ensuing cord compression. This case stresses the rapidity of development of paralysis or other neurologic complications, as well as the difficulty and emergent nature of the diagnosis of epidural abscess in this situation.

High-dose cyclophosphamide and intermediate-dose methotrexate for the treatment of far-advanced Burkitt's lymphoma.

Ten consecutive patients were entered on a pilot study--for the treatment of children with far-advanced Burkitt's lymphoma--which was conducted at the Roswell Park Memorial Institute (R.P.M.I.) and the University of Rochester Medical Center (U. of R.). With the use of high-dose cyclophosphamide (HDC), intermediate-dose methotrexate (IDM), vincristine (VCR), prednisone (PRED), and intrathecal (IT) chemotherapy, nine of these 10 children achieved a complete remission (CR), and six of these nine children have remained in CR for more than 3 years and are likely cured of their disease. This regimen had significant, but generally manageable, toxicity. A single patient with ataxia-telangiectasia died during induction therapy of pneumonia and a gastrointestinal hemorrhage. A single patient with severe hemorrhagic cystitis required the substitution of cytosine arabinoside for cyclophosphamide (Cytoxan) in the regimen. One of five children presenting with bone marrow involvement survives. Such involvement appears to place patients at greater risk for developing meningeal disease. Attempts at more aggressive methods of central nervous system (CNS) prophylaxis should be employed in this group of patients.