Serum levels of lactoferrin and myeloperoxidase in chronic idiopathic and secondary neutropenia. A preliminary report

In 20 patients with chronic neutropenia, serum lactoferrin (S-LF) and serum myeloperoxidase (S-MPO) levels were assessed. By immunofluorescence, granulocyte-bound immunoglobulins were detected in 12 patients, whereas circulating immune complexes were found in the blood of 8 patients by the 125-I-C1q-binding test (C1q-BT). In both groups of patients, there was a relative increase of S-LF and a relative or sometimes absolute increase of S-MPO. In the latter group, results of the C1q-BT correlated positively with S-MPO but negatively with neutrophil counts. No correlations between S-LF or S-MPO and the results of the granulocyte immunofluorescence test were found. Our results suggest that S-LF and S-MPO levels may be helpful in the further study of patients with chronic neutropenia, to gain more insight into the pathogenetic mechanisms operative in this disease.     

Serum granulocyte colony-stimulating factor levels in patients with chronic neutropenia of childhood: modulation of G-CSF levels by myeloid precursor cell mass

The serum G-CSF levels of eight patients with severe congenital neutropenia (SCN) were found to be significantly higher than those of 22 patients with chronic benign neutropenia (CBN). The relative number of cells expressing the G-CSF receptor in light density bone marrow cells (LDBMC) was lower in patients with SCN than in patients with CBN or in normal subjects. When recombinant human G-CSF was incubated with LDBMC, G-CSF levels were decreased by LDBMC from normal subjects and CBN patients, but not by those from SCN patients. Serum G-CSF concentrations, which are affected by mature neutrophils, may also be modulated by myeloid precursor cells in the bone marrow.     

Serum Myeloperoxidase and Lactoferrin in Neutropenia

Radioimmunosorbent assays for determination of serum content of the neutrophil proteins myeloperoxidase and lactoferrin are described. Serial studies were performed in patients with neutropenia. In 2 cases of cyclic neutropenia the myeloperoxidase level showed slight variations within the normal range during the cycle while lactoferrin displayed a clear correlation with neutrophil counts. In 1 case with persistent agranulocytosis myeloperoxidase was normal but lactoferrin was extremely low. During the regeneration phase of drug-induced neutropenia neutrophil counts and serum lactoferrin increased in a parallel fashion. Since serum myeloperoxidase was normal during profounded neutropenia it is suggested to derive primarily from myeloperoxidase-rich granulopoietic precursor cells of the marrow. Serum lactoferrin on the other hand seems to derive from leakage of more mature granulopoietic cells of blood and marrow. Studies of neutrophil proteins of serum may aid in evaluation of neutropenic patients.     

Serum and plasma measurements of neutrophil granule proteins during cardiopulmonary bypass: a model to estimate human turnover of lactoferrin and myeloperoxidase

During extracorporeal circulation (ECC), granule proteins such as lactoferrin and myeloperoxidase escape to the plasma in large amounts. ECC was used to study the turnover of these proteins and compare their variations in plasma and serum. During ECC both proteins rose to very high levels and the variations were similar in both serum and plasma (r = 0.93 for lactoferrin and 0.80 for myeloperoxidase). The initial elimination after termination of operation (t 1/2) was, for lactoferrin 0.75 and for myeloperoxidase 0.5 h. Both proteins followed a second order kinetics of elimination with a t 1/2 for lactoferrin of about 9 and for myeloperoxidase of about 25 h. 48 and 72 h postoperatively serum levels, but not plasma levels, of myeloperoxidase rose again 10-fold. We conclude that serum and plasma measurements of neutrophil granule proteins are complementary. The period after disconnection of the patient from the extracorporeal device may be used to estimate the turnover kinetics of neutrophil granule proteins.     

Serum myeloperoxidase and lactoferrin in neutropenia.

Radioimmunosorbent assays for determination of serum content of the neutrophil proteins myeloperoxidase and lactoferrin are described. Serial studies were performed in patients with neutropenia. In 2 cases of cyclic neutropenia the myeloperoxidase level showed slight variations within the normal range during the cycle while lactoferrin displayed a clear correlation with neutrophil counts. In 1 case with persistent agranulocytosis myeloperoxidase was normal but lactoferrin was extremely low. During the regeneration phase of drug-induced neutropenia neutrophil counts and serum lactoferrin increased in a parallel fashion. Since serum myeloperoxidase was normal during profounded neutropenia it is suggested to derive primarily from myeloperoxidase-rich granulopoietic precursor cells of the marrow. Serum lactoferrin on the other hand seems to derive from leakage of more granulopoietic cells of blood and marrow. Studies of neutrophil proteins of serum may aid in evaluation of neutropenic patients.     

Increased levels of soluble flt-3 ligand in serum and long-term bone marrow culture supernatants in patients with chronic idiopathic neutropenia

The levels of serum and long-term bone marrow culture supernatant soluble flt-3 ligand (sFL) were determined in 54 patients with chronic idiopathic neutropenia (CIN) and 16 normal controls. Both serum and supernatant sFL levels were significantly increased in the patients compared with controls. Individual sFL values inversely correlated with the number of circulating neutrophils and the proportion of bone marrow CD34+ cells. Supernatant sFL values positively correlated with the levels of supernatant G-CSF. These findings suggest that the impaired myelopoiesis in CIN patients is accompanied by a compensatory mechanism attempting to increase the neutrophil production at the myeloid progenitor cell level.     

Fecal lactoferrin, myeloperoxidase and serum C-reactive are effective biomarkers in the assessment of disease activity and severity in patients with idiopathic ulcerative colitis

Background and Aim:  Disease activity and severity of ulcerative colitis (UC) is assessed using colonoscopy, which is invasive, costly and has poor patient acceptability. The role of non-invasive biomarkers of intestinal inflammation in the evaluation of patients with UC is not known. The aim of the study was to examine the role of serum C-reactive protein (SCRP), fecal myeloperoxidase (FMPO) and fecal lactoferrin (FLF) in assessing disease severity, activity and response to therapy.
Methods:  Consecutive patients with idiopathic UC (IUC) attending our hospital from July 2005 to September 2006 were studied. All underwent clinical, endoscopic and histological assessment for disease activity, extent, severity and estimation of SCRP, FMPO and FLF levels at baseline and follow up (FU). An equal number of healthy age-matched controls were studied for biomarker levels.
Results:  A total of 37 patients (mean age 37 ± 12 years) were studied. All three biomarkers were elevated more often in the cases than in the controls (all P  = 0.000). Cases with severe IUC had higher CRP, MPO and FLF titers than those without severe IUC. At FU, a significant fall in biomarker levels paralleled the reduction in Mayo's scores. All three biomarkers showed a high degree of correlation with each other. The areas under the curve for FLF, MPO and CRP were 1.00, 0.867 and 0.622, respectively. The sensitivity and specificity of markers were: FLF (94%, 100%), FMPO (89%, 51%) and SCRP (24%, 100%).
Conclusion:  Biomarkers are useful in assessing disease activity, severity and response to therapy in patients with UC. They showed a high degree of correlation with each other.     

Diagnostic and prognostic significance of serum measurements of lactoferrin, lysozyme and myeloperoxidase in acute myeloid leukemia (AML): recognition of a new variant, high-lactoferrin AML

92 patients with acute myeloid leukemia were classified according to the FAB classification (M1 n = 20, M2 n = 43, M3 n = 1, M4 n = 19, M5a n = 2, M5b n = 2, and M6 n = 5 patients). Serum measurements of lactoferrin (LF), myeloperoxidase (MPO) and lysozyme (LYS) were performed before the start of treatment. LF was significantly lower in M1 when compared with M2 but not as compared to M4, MPO was significantly higher in M2 and M4 than in M1, but comparable MPO levels were found in M2 and M4. LYS was significantly elevated in M2 in comparison with M1, and in M4 when compared to both M1 and M2. Polymorphonuclear granulocytes (PMNs) in M1 were significantly reduced when compared with M2 and M4, whereas mononuclear cells were significantly increased in M4 in comparison with both M1 and M2. FAB classification did not generate any prognostic information. When the patients were, instead, subdivided according to LF levels were found prognostically significant differences. Of patients below 100 micrograms/l, 44% went into remission as compared to 77% with LF from 101 to 400 micrograms/l. In patients with LF levels above 400 micrograms/l the remission frequency was only 14%. Multivariate statistical analysis on the data further suggested that lactoferrin may be used as an independent prognostic indicator. We conclude that although determination of the serum-levels of lactoferrin, lysozyme and myeloperoxidase in certain cases may be valuable as a supplement to the morphological examination of acute myeloid leukemia, it is evident that none of the three determinations can be used alone to distinguish between the FAB groups.     

Myeloid regeneration after bone-marrow transplantation monitored by serum measurements of myeloperoxidase, lysozyme and lactoferrin

Bone-marrow regeneration after chemo- and radiotherapy-induced aplasia can be monitored by serum levels of myeloperoxidase (MPO), lysozyme (LYS) and lactoferrin (LF). In 10 patients with leukemia, serum measurements were performed before and after bone-marrow transplantation. Bone-marrow regeneration was suggested by increments in serum MPO and LYS 5 and 4 days prior to the increase in mononuclear cells (Mono) and 10 and 9 d before the increase in polymorphonuclear leukocytes (PMN) in the peripheral blood. LF started to rise 4.5 d before detectable circulating PMNs. 2 patients with early relapses of leukemia post transplantation are shown to display atypical patterns of serum MPO and LYS. We conclude that serum measurements of MPO, LYS and LF may be used as early and sensitive means to monitor bone-marrow activity during hematological regeneration. However, the findings also strongly support the earlier proposal that MPO alone may be used to reflect myeloid activity in the bone-marrow in general.     

Non-immune chronic idiopathic neutropenia of adult: an overview.

There is strong evidence that non-immune chronic idiopathic neutropenia of adult is a cytokine-mediated syndrome characterized by (a) neutropenia of varying degree associated with a low number of lineage-specific CD34+ cells and increased production of inhibitors of hematopoiesis, including transforming growth factor-beta1 and tumor necrosis factor-alpha; (b) lymphopenia due to selective loss of primed/memory T-cells and NK cells; (c) increased splenic volume on ultrasonography in 48.1% of patients; (d) osteopenia and/or osteoporosis in 60.0% of patients; (e) anemia, mostly of the type of anemia of chronic disease, in 15.6% of patients; (f) features of chronic antigenic stimulation, including increased proportion of bone marrow plasma cells, increased serum levels of IgG1 and/or IgA, increased frequency of monoclonal gammopathy of undetermined significance, increased frequency of antinuclear antibodies with specific reactivity, and increased serum levels of circulating immune complexes; and (g) increased concentrations of a variety of macrophage-derived pro-inflammatory cytokines and chemokines capable of affecting bone metabolism, bone marrow function, and leukocyte trafficking. All these findings are suggestive of the existence of an unrecognized low-grade chronic inflammatory process which may be involved in the pathogenesis of the disorder. Neutropenia in these patients is probably the result of a combination of at least three factors, reduced neutrophil production in bone marrow, enhanced neutrophil extravasation, and increased sequestration and/or extravasation of neutrophils into the spleen.     

Activated T-lymphocytes with myelosuppressive properties in patients with chronic idiopathic neutropenia

To characterize the cellular components responsible for the impaired granulopoiesis in chronic idiopathic neutropenia (CIN), we investigated the origin of the proapoptotic cytokine producing cells in the bone marrow (BM) microenvironment of CIN patients. We found that the interferon gamma (IFN gamma) and/or Fas-ligand expressing cells in patient BM mononuclear cells and long-term BM culture stroma cells were the CD3(+) T-lymphocytes but not the CD14(+) monocytes/macrophages. The percentage of activated T-lymphocytes was increased in patients' BM as indicated by the proportions of human leucocyte antigen (HLA)-DR(+), CD25(+), CD38(+), CD69(+) and Fas(+) cells within the CD3(+) fraction. Intracellular IFN gamma expression was higher in the BM than peripheral blood of the patients and was associated with increased BM T-lymphocyte numbers. In crossover experiments, patient CD3(+) T-lymphocytes conferred autologous and allogeneic haemopoietic progenitor cell colony inhibition. Patients' T-cell receptor repertoire and polymerase chain reaction analysis did not reveal any clonal T-lymphocyte expansion, suggesting the absence of a direct, antigen-driven recognition of CD34(+) myeloid progenitor cells by patient T-lymphocytes. We conclude that CIN patients have increased number of activated T-lymphocytes in the BM, probably in the setting of a localized polyclonal immune reaction and that these cells confer an inhibitory effect on myelopoiesis through myelosuppressive cytokines including Fas-ligand and IFN gamma.     

Patients with non‐immune chronic idiopathic neutropenia syndrome have increased splenic volume on ultrasonography

Clinically detectable splenomegaly is rarely seen in patients with non‐immune chronic idiopathic neutropenia syndrome (NI‐CINS). Using ultrasound, we estimated splenic volume in 52 NI‐CINS patients and 14 age‐ and sex‐matched normal controls by determining the ‘corrected splenic index’ (CSI) from the product of length, width and thickness of the organ expressed in cm³/m² body surface area. We found that CSI was significantly higher in the group of patients compared to controls (202.8 ± 82.0 vs. 133.8 ± 28.1 cm³/m², P=0.003), and that individual CSI values was inversely correlated with the number of circulating neutrophils (r=–0.5097, P < 0.0001). About 48.1% of the patients had CSI above 190 cm³/m² body surface, representing the upper 95% confidence limit of values found in the controls. Patients also had increased serum concentrations of pro‐inflammatory cytokines and chemokines mainly produced by activated macrophages (IL‐1β, TNF‐α, RANTES and IL‐8), as well as increased serum levels of soluble cell adhesion molecules derived from activated endothelium (sE‐Selectin, sICAM and sVCAM). We hypothesize that the increased splenic volume in NI‐CINS patients may be due to the accumulation of activated macrophages inside the spleen, possibly as the result of an unrecognized low‐grade chronic inflammatory process. The nature of such an inflammation is unknown. A study was designed to search for viral or bacterial genomic material in patients’ bone marrow stromal macrophages in which the unknown causal agent might be located.     

成人慢性特发性中性粒细胞减少症的病因探讨

目的：探讨成人慢性特发性中性粒细胞减少症（ACIN）的发病机制。方法：分析61例ACIN患者的临床及实验资料。结果：ACIN患者骨髓粒细胞成熟比值，浆细胞比例、组织细胞比例升高，血清白细胞介素－6、白细胞介素－8水平升高，抗核抗体阳性率升高，与正常对照比较差异均有极显著性意义。结论：持续低程度的慢性炎症反应可能与ACIN的发病有关。     

Successful treatment of chronic severe neutropenia with weekly recombinant granulcyte-colony stimulating factor

Daily treatment for symptomatic chronic neutropenia with recombinant granulocyte-colony stimulating factor (rhG-CSF) filgrastim is costly and sometimes causes neutrophillia. We report the use of weekly filgrastim in a 40-year-old man with life-long symptomatic neutropenia. Baseline neutrophil counts were <1×10⁹/l 60% of the time, and fell below 0.5×10⁹/l for 7 d periods every 22 d. Following 1 year of weekly filgrastim treatment, the absolute neutrophil count was maintained >1×10⁹/l (averaging 2×10⁹/l) and the frequency and severity of symptoms were reduced by 85%. Therefore the benefits of filgrastim for the treatment of at least one form of chronic severe neutropenia can be derived from weekly rather than daily doses.     

Two case studies of chronic idiopathic neutropenia preceding acute myeloid leukaemia

Chronic idiopathic neutropenia is regarded as a benign disorder without risk of malignant transformation. We present two patients with chronic idiopathic neutropenia who showed disease progression to acute myeloid leukaemia. Sequence analysis of the granulocyte-colony stimulating factor receptor (G-CSFR) gene from leukaemic DNA did not reveal any mutations and microsatellite analysis provided no evidence of microsatellite instability or loss of constitutional heterozygosity. These case studies suggest that chronic idiopathic neutropenia may constitute a preleukaemic condition in some patients. Alterations of the G-CSFR or defective DNA mismatch repair do not appear to be involved in malignant transformation.     

Pro-inflammatory bone marrow milieu in patients with chronic idiopathic neutropenia is associated with impaired local production of interleukin-10

The levels of interleukin-10 (IL-10) were evaluated in long-term bone marrow (BM) culture supernatants from 54 patients with chronic idiopathic neutropenia (CIN) and 30 healthy volunteers using enzyme-linked immunoabsorbent assay. Cytokine levels were significantly reduced in patients, compared with controls, and strongly correlated with peripheral blood neutrophil counts. Low levels of supernatant IL-10 were associated with increased values of supernatant IL-1beta, tumour necrosis factor-alpha, IL-6 and transforming growth factor-beta(1). We suggest that the pro-inflammatory milieu in the BM of CIN patients may be causatively related to the impaired production of IL-10, a cytokine normally displaying strong anti-inflammatory properties.     

Helicobacter pylori infection is probably the cause of chronic idiopathic neutropenia (CIN)-associated splenomegaly

Splenic volume and Helicobacter pylori (H. pylori) infection were evaluated in 67 patients with chronic idiopathic neutropenia (CIN) and 39 healthy individuals. Using ultrasound, splenomegaly was found in 61.7% of H. pylori-infected subjects compared to only 8.7% noted in the group of H. pylori-non-infected individuals (P < 0.0001). Splenomegaly was also found in 47.8% of CIN patients compared to 12.8% in the group of non-CIN subjects (P = 0.0003). However, applying the two-way ANOVA test, a statistically significant effect on splenic volume was documented for "factor H. pylori " (F1(102) = 16.800, P < 0.0001) but not for "factor CIN" (F1(102) = 3.213, P = 0.0760), suggesting that CIN-associated splenomegaly is probably due to H. pylori infection.     

Increased in vitro neutrophil adherence in a case of chronic idiopathic neutropenia

Summary In this report we describe a patient with persistent neutropenia whose neutrophils showed increased adhesion in a microplate assay. In three separate assays, from 12·5% to 13·7% of the patient's blood neutrophils exhibited spontaneous (unstimulated) adhesion to fetal bovine serum-coated microplate wells, much higher than adhesion of cells from healthy controls (1·9% 2·5 SD, n = 20). The difference of spontaneous adhesion between the patient's and control neutrophils was even higher when cells from a skin-window exudate were examined (patient: 42·1–100% adhesion: control: 3·6% 3·5 SD, n = 20). Over 80% inhibition of the increased adhesion was produced by the 60.3 anti-CD 18 monoclonal antibody, suggesting an involvement of β2-integrins.