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.     

Pneumococcal infections after human bone-marrow transplantation.

Seven of 26 long-term survivors (greater than 7 months post-transplant) of bone-marrow transplantation developed penicillin-sensitive pneumococcal infections more than 7 months after transplantation. One patient had two infections. Six of eight infections were associated with pneumococcal bacteremia, and Streptococcus pneumoniae type 6A was isolated in three cases. Two infections were fatal. All patients had normal nematopoietic function, and none was receiving immunosuppressive therapy. The development of pneumococcal infection was significantly associated with males and with abnormally low or high serum IGG and IgM levels but not with graft-versus-host disease. Serum opsonic activity for S. pneumoniae type 6A was decreased in six of the seven patients when compared to normal pooled serum in an in-vitro bactericidal assay. Four of the six patients with impaired opsonic activity had low serum antibody levels for S. pneumoniae type 6A capsular polysaccharide, while the other two patients had low serum CH100 complement activity. Bone-marrow transplant recipients have an increased susceptibility to pneumococcal infections and should be evaluated for prophylactic penicillin or pneumococcal vaccination.     

β2-Microglobulin, lysozyme and lactoferrin in cerebrospinal fluid in patients with lymphoma or leukaemia: relationship to CNS involvement and the effect of prophylactic intrathecal treatment with methotrexate

Central nervous system (CNS) involvement in patients with leukaemia or lymphoma presents a diagnostic problem. This study was conducted to test whether combined measurements of various cellular markers such as beta 2-microglobulin (beta 2m), lactoferrin (LF) and lysozyme (LYS) in the cerebrospinal fluid (CSF) might aid in the diagnosis of CNS involvement in such patients. Forty-two patients were studied. Sixteen were considered to have CNS involvement and 26 showed no signs of such involvement. In the group with symptoms or signs of CNS involvement, nine patients out of 12 had increased total protein in CSF, 14 of 14 increased beta 2m, 14 of 16 increased LYS and five of 15 increased LF. In patients without CNS involvement total protein was increased in four of 25, beta 2m in three of 21, LYS in four of 28 and LF in one of 28 patients. The differences were statistically significant (P less than 0.01, P less than 0.001, P less than 0.001 and P less than 0.05, respectively). Prophylactic intrathecal methotrexate treatment in patients with acute lymphoblastic leukaemia caused an increase in the CSF of beta 2m, LYS and LF but not of total protein, which may reflect a drug-induced inflammatory reaction in the CNS. We conclude that combined measurements of the three cell markers add to our understanding of the cellular reaction to malignant cells in the CNS in leukaemia and lymphoma and may be valuable supplements in the diagnosis of this CNS involvement.     

The effect of rGM-CSF on neutrophil and eosinophil regeneration after ABMT as monitored by circulating levels of granule proteins

Summary. In order to further evaluate the effects of rGM-CSF on the reconstituting granulopoiesis, plasma and serum levels of myeloperoxidase (MPO) and lactoferrin (LF), as well as serum levels of eosinophil cationic protein (ECP), were monitored daily during a period of 3–4 weeks following ABMT in a group of 22 patients treated with either rGM-CSF ( n =11) or placebo ( n =11). Despite faster increase in the neutrophil counts in the rGM-CSF group, we did not observe any difference either in P-MPO or in P-LF during the period of early engraftment (days 11–19). This finding indicates that the proliferative effect of rGM-CSF on the neutropoiesis may be overestimated when neutrophil counts alone are taken into consideration, and suggests that other mechanisms may have contributed to the increase in the number of circulating neutrophils. The ratio of the serum to plasma level of LF, but not of MPO, was higher in the rGM-CSF group, probably reflecting a specific in vivo neutrophil priming effect. In the rGM-CSF group there was a clear increase of S-ECP during the second and third week post transplant, corresponding to an increase in eosinophil counts, which indicates that rGM-CSF stimulated eosinophil reconstitution without causing excessive activation of the mature eosinophils.     

Bone-marrow regeneration after therapy-induced hypoplasia monitored by serum measurements of lactoferrin, lysozyme and myeloperoxidase

Serum levels of lactoferrin, lysozyme and myeloperoxidase were measured sequentially after induction treatment in 30 patients with AML in order to test the hypothesis that these proteins may be used to monitor activity and different stages of myelopoiesis in the bone-marrow. The results showed that myeloperoxidase and lysozyme started to rise again 6-8 days after initiation of treatment as compared to 12 d for lactoferrin and 16 d for blood polymorphonuclear leukocytes. The rate of marrow regeneration was exponential and estimated to be 9-20% per d. The comparison between two different chemotherapy regimes showed that the initial reduction in lysozyme, in contrast to other measured variables, was significantly larger with the therapy that contained vincristine and glucocorticosteroids. This might reflect a reduction in lysozyme secretion in addition to the effects on the leukemic cell mass. We conclude that the measurements of lactoferrin, lysozyme and myeloperoxidase in serum under certain circumstances may be used to monitor the myelopoietic activity in the bone-marrow.     

Ultrastructural localization of lactoferrin and myeloperoxidase in human neutrophils by immunogold

Colloidal gold was used as a marker for immunoelectron microscopy to localize lactoferrin (LF) and myeloperoxidase (MPO) in human peripheral blood neutrophils. Cells were reacted with monospecific antibodies against LF or MPO and then with gold-labeled antiglobulin. MPO cytochemistry was also associated with immunologic detection of LF. Immunologic labeling of thin sections after embedding in glycol methacrylate gave good ultrastructural morphology and specific localization of both proteins. MPO was detected in the large azurophil granules, whereas LF was consistently localized in the matrix of another population of morphologically distinct granules, smaller and more numerous than azurophil granules. When cytochemical detection of MPO was coupled with immunologic detection of LF, LF was observed in the population of MPO-negative granules, which were identified as specific. This was confirmed on cells that were permeabilized with saponin and stained for LF and MPO before embedding. No other neutrophil organelles displayed labeling for LF; other blood cells also were unreactive for LF. In the bone marrow, myeloblast and promyelocyte granulations were not stained and LF-containing granules appeared at the myelocyte stage. In conclusion, we confirm previous biochemical and light microscopic studies by ultrastructural demonstration of LF and MPO in two categories of granules, the specific and azurophil granules, respectively. The method described in this article avoids disruption caused by cell fractionation procedures. In the future, other intragranular proteins can be localized by a similar approach.     

Granule protein contents of polymorphonuclear leucocytes and serum in 30 cases of myelodysplastic syndromes]

Intracellular contents and serum levels of neutrophilic granule protein components, including myeloperoxidase (MPO), lactoferrin (LF) and lysozyme (LYZ), were investigated in 30 cases of myelodysplastic syndromes (MDS), with 59 healthy adult donors as controls. Both neutrophilic MPO and LF decreased significantly, suggesting the transformation of abnormal clones. Both serum levels of LF and LYZ exhibited a considerable fluctuation which may reflect reflect granulopoiesis in the bone marrow. We are of the opinion that measurement of intracellular MPO, LF, LYZ and their serum levels may aid in the diagnosis and prognosis of MDS and is proved to be of great clinical significance.     

The effect of granulocyte colony-stimulating factor (G-CSF) on the degranulation of secondary granule proteins from human neutrophils in vivo may be indirect

Granulocyte colony-stimulating factor (G-CSF) was administered at a dose of 7.5 or 10 μg/kg s.c. once daily for 6 d (days 1–6) to two groups consisting of eight and six healthy volunteers. The administration of G-CSF resulted in a rapid decrease in neutrophil counts and serum levels of the secondary granule protein, human neutrophil lipocalin (HNL) after 30 min, followed by a recovery and gradual increase within 180 min. The number of circulating neutrophils and plasma and serum levels of neutrophil secondary granule proteins were dramatically elevated on day 2 (1 d after the administration of G-CSF) and stayed so until day 7. The plasma levels of HNL and lactoferrin (LF) showed a biphasic pattern with peaks at day 2 and days 5–7, and remained highly elevated at day 12. The serum levels of HNL and LF increased rapidly (about 8-fold and 6-fold, respectively) on day 2 and stayed elevated until day 7, subsequently returning to baseline levels. At day 5, neutrophil release induced in vitro by f-MLP was significantly enhanced. The cellular contents of HNL and LF were reduced to about 50% of levels before G-CSF administration at day 5. The release of lactoferrin and HNL, but not of myeloperoxidase (MPO), was slightly enhanced after preincubation of isolated normal neutrophils with G-CSF in vitro, but no obvious release of these proteins was observed with G-CSF alone. The administration of G-CSF resulted in a dramatic increase in the alkaline phosphatase (AP) activity in the plasma membrane, with maximal activity occurring at day 5. Furthermore, during administration of G-CSF, TNF-α in plasma increased about 25-fold. TNF-α started to rise at day 2 and peaked at day 6. After discontinuation of G-CSF the levels of TNF-α gradually decreased. The elevated levels of TNF-α (tumour necrosis factor-α) were temporally correlated to the other signs of neutrophil activation. GM-CSF and IL-8, however, were not detected in plasma. Our data suggest that G-CSF affects the neutrophils not only directly but also indirectly by the induction of the production of other cytokines such as TNF-α.     

Anti-neutrophil cytoplasmic antibody in patients with systemic lupus erythematosus is unrelated to clinical features

Summary To investigate the incidence, the specificity and clinical significance of positivity for serum anti-neutrophil cytoplasmic antibody (ANCA) in 31 patients with systemic lupus erythematosus (SLE), the indirect immunofluorescence (IIF) technique and enzyme-linked immunosorbent assay (ELISA) were used to measure ANCA. Purified myeloperoxidase (MPO), lactoferrin (LF), cathepsin-G (CTG) and elastase (HLE) served as ANCA antigens for ELISA. Thirteen (42%) of the 31 SLE patients showed positivity for perinuclear, but not cytoplasmic, ANCA by IIF. Five of 31 sera were positive for MPO, 10 for LF, 1 for CTG and 0 for HLE by ELISA. Patients positive for ANCA had a higher score of SLE disease activity index (SLEDAI) than those without ANCA. There was no correlation between ANCA positivity, clinical manifestations, or organic involvement. While the ANCA in patients with SLE reflected disease activity, it was unrelated to organic involvement.     

Oral N-acetylcysteine reduces selected humoral markers of inflammatory cell activity in BAL fluid from healthy smokers: correlation to effects on cellular variables

Bronchoalveolar lavage (BAL) was performed on eleven healthy smokers before and after eight weeks of oral treatment with N-acetylcysteine (NAC) 200 mg t.i.d. The concentrations of selected eosinophil and neutrophil granule constituents and of selected proteases and protease inhibitors, albumin and endotoxin were determined in the recovered BAL fluid and in plasma or serum samples. In addition, in vitro chemotactic activities for neutrophils and eosinophils were assessed in the BAL fluid. Significant reductions in BAL fluid content of lactoferrin (LF), eosinophil cationic protein (ECP), antichymotrypsin (ACT) and chemotactic activity for neutrophils were recorded after NAC treatment. The levels of other examined markers tended to be reduced or were not affected. In serum/plasma, the concentrations of myeloperoxidase (MPO) and elastase were reduced after NAC treatment whereas concentrations of other constituents examined were unaltered. These data, together with previously reported findings, suggest that oral NAC may influence the activity of "inflammatory" cells in the bronchoalveolar space of smokers.     

Two serologic markers to monitor the engraftment, growth, and treatment response of human leukemias in severe combined immunodeficient mice

We have investigated human lactate dehydrogenase (LDH) isoenzymes and human nuclear matrix protein 41/7 (NMP 41/7) as potential serologic markers to monitor the course of human leukemia in severe combined immunodeficient (SCID) mice. Following the transplantation of 10(6) human acute lymphoblastic leukemia (ALL) Nalm-6 cells, human specific LDH isoenzymes were measurable in the serum of SCID mice as early as 7 days after transplantation, although serum total LDH increased in some animals as early as 5 days after transplantation. Human NMP 41/7 was measurable in all animals at day 15 after leukemia cell injection. Serum levels of total LDH, human specific LDH and NMP 41/7 increased progressively over time, reaching total LDH levels as high as 50,000 U/L at day 25 after transplantation. To determine whether the levels of LDH and NMP 41/7 in serum were a reflection of human tumor burden, we studied these serologic markers in SCID mice bearing measurable subcutaneous human neuroblastoma tumors, or compared the serum levels of these markers with the number of human leukemia CD10+ cells in the bone marrow of the SCID mice. The serum levels of total LDH, human specific LDH isoenzymes, and NMP 41/7 correlated well with tumor burden, and they drastically decreased or disappeared from serum after the human leukemia or neuroblastoma cells were selectively killed with a single intravenous (IV) injection of 1 to 3 micrograms diphtheria toxin (DT) (the cellular receptor for DT is present on human cells, but not on mouse cells). Paraplegic mice with central nervous system leukemia completely recovered after DT treatment. We conclude that measurements of serum levels of total LDH, human LDH isoenzymes, and NMP 41/7 are sensitive, quantitative, rapid, and easy to perform serologic methods useful to monitor the engraftment, progression, and treatment response of human leukemia in SCID mice.     

髓过氧化物酶活性升高在急性冠状动脉综合征患者中的临床意义

目的探讨急性冠状动脉综合征(ACS)患者血清髓过氧化物酶(MPO)活性变化的临床意义。方法采用比色法测定实验组和对照组血清MPO活性。结果(1)ACS包括不稳定型心绞痛/无ST段抬高的心肌梗死(UAP/NSTEMI)和ST段抬高的心肌梗死(STEMI)患者血清MPO活性明显升高,与正常对照组(Control组)、稳定型心绞痛(SAP)组比较有显著性差异(P<0.001)。STEMI患者血清MPO活性高于UAP/NSTEMI(P<0.001)。(2)SAP血清MPO活性高于Control组(P<0.001)。(3)ACS患者接受经皮冠状动脉介入治疗(PCI)术后MPO活性较术前显著升高(P<0.001)。(4)ACS患者血清MPO活性与白细胞计数(WBC)呈正相关(r=0.619,P=0.001),与年龄、性别、高敏感C反应蛋白、总胆固醇、低密度脂蛋白、高密度脂蛋白、三酰甘油无相关性。结论血清MPO活性升高可能与ACS的发生有关。MPO是一种预测ACS的炎症标志物。     

Ischemic preconditioning decreases C-X-C chemokine expression and neutrophil accumulation early after liver transplantation in rats

AIM: Polymorphonuclear neutrophil (PMN) plays a major role in liver ischemia/reperfusion injury. Protective effect of ischemic preconditioning (IP) has been confirmed in liver ischemia/reperfusion injury. The purpose of this study was to investigate the effect of IP on C-X-C chemokine expression and PMNs recruitment eady after liver transplantation.METHODS: Male Sprague-Dawley rats were used as donors and recipients of orthotopic liver transplantation (OLT). The donor liver was stored 24 hours in University of Wisconsin(UW) solution at 4 ℃ pre-implantation, IP was done by damp of the portal vein and hepatic artery of the donor liver for 10 minutes followed by reperfusion for 10 minutes before harvesting. The neutrophilic infiltration in liver was quantified using a myeloperoxidase (MPO) assay. Intragrafl: expression of macrophage inflammatory protein-2 (MIP-2) mRNA was investigated with in situ hybridization, The serum levels of MIP-2 and tumor necrosis factor (TNF)-α were also monitored,RESULTS: After liver transplantation without IP, the hepatic MPO increased significantly compared with sham operated group. In IP group, PMN in liver indicated by MPO was reduced significantly. In situ hybridization showed no MIP-2 mRNA in sham group but dramatic expression in hepatocytes in non-IP group. In IP group, MIP-2 mRNA was significantly down-regulated. Similarly, serum MIP-2 and TNF-α levels were significantly elevated in non-IP group and both were reduced in IP group.CONCLUSION: IP might protect graft liver from preservation-reperfusion injury after OLT through down-regulating C-X-C chemokine expression of hepatocytes, and alleviating PMNs recruitment after reperfusion.     

Ischemic preconditioning decreases C-X-C chemokine expression and neutrophil accumulation early after liver transplantation in rats

AIM: Polymorphonuclear neutrophil (PMN) plays a major role in liver ischemia/reperfusion injury. Protective effect of ischemic preconditioning (IP) has been confirmed in liver ischemia/reperfusion injury. The purpose of this study was to investigate the effect of IP on C-X-C chemokine expression and PMNs recruitment early after liver transplantation.METHODS: Male Sprague-Dawley rats were used as donors and recipients of orthotopic liver transplantation (OLT). The donor liver was stored 24 hours in University of Wisconsin (UW) solution at 4℃ pre-implantation. IP was done by clamp of the portal vein and hepatic artery of the donor liver for 10minutes followed by reperfusion for 10 minutes before harvesting. The neutrophilic infiltration in liver was quantified using a myeloperoxidase (MPO) assay. Intragraft expression of macrophage inflammatory protein-2 (MIP-2) mRNA was investigated with in situ hybridization. The serum levels of MIP-2 and tumor necrosis factor (TNF)-α were also monitored.RESULTS: After liver transplantation without IP, the hepatic MPO increased significantly compared with sham operated group. In IP group, PMN in liver indicated by MPO was reduced significantly. In situ hybridization showed no MIP2 mRNA in sham group but dramatic expression in hepatocytes in non-IP group. In IP group, MIP-2 mRNA was significantly down-regulated. Similarly, serum MTP-2 and TNF-α levels were significantly elevated in non-TP group and both were reduced in IP group.CONCLUSION: IP might protect graft liver from preservationreperfusion injury after OLT through down-regulating C-X-C chemokine expression of hepatocytes, and alleviating PMNs recruitment after reperfusion.     

Anti-inflammatory effects of Mangifera indica L. extract in a model of colitis

AIM:To investigate the effect of aqueous extract from Mangifera indica L.(MIE)on dextran sulfate sodium (DSS)-induced colitis in rats.METHODS:MIE(150 mg/kg)was administered in two different protocols:(1)rectally,over 7 d at the same time as DSS administration;and(2)once daily over 14 d (by oral gavage,7 d before starting DSS,and rectally for 7 d during DSS administration).General observations of clinical signs were performed.Anti-inflammatory activity of MIE was assessed by myeloperoxidase(MPO)activity. Colonic lipid peroxidation was determined by measuring the levels of thiobarbituric acid reactive substances (TBARS).Reduced glutathione(GSH)levels,expression of inflammatory related mediators[inducible isoforms of nitric oxide synthase(iNOS)and cyclooxygenase (COX)-2,respectively]and cytokines[tumor necrosis factor(TNF)-αand TNF receptors 1 and 2]in colonic tissue were also assessed.Interleukin(IL)-6 and TNF-α serum levels were also measured. RESULTS:The results demonstrated that MIE has anti-inflammatory properties by improvement of clinical signs,reduction of ulceration and reduced MPO activity when administered before DSS.In addition,administration of MIE for 14 d resulted in an increase in GSH and reduction of TBARS levels and iNOS,COX-2, TNF-αand TNF R-2 expression in colonic tissue,and a decrease in IL-6 and TNF-αserum levels. CONCLUSION:MIE has anti-inflammatory activity in a DSS-induced rat colitis model and preventive administration(prior to DSS)seems to be a more effective protocol.     

Serum transcobalamin levels as an early indicator of bone marrow engraftment following transplantation

Three B12 binding proteins, the transcobalamins TCI, TCII and TCIII, were determined serially in the serum of five patients who underwent bone marrow transplantation. The increase in TCII, followed by the increase in TCI, proved to be an early indicator of bone marrow regeneration, reaching a peak of up to twice its normal levels at least 5 d prior to the rise of the peripheral white blood cell count.     

Reduced risk of recurrent leukaemia in bone marrow transplant recipients after cytomegalovirus infection

The first 72 consecutive bone-marrow transplant recipients with haematological malignancies (29 with acute nonlymphoblastic leukaemia, 31 with acute lymphoblastic leukaemia, nine with CML and three with myelofibrosis, IgA myeloma and T-cell lymphoma, respectively) were investigated for the frequency of relapses 1 year or later after bone-marrow transplantation. Seven relapses occurred from 30 to 850 d after transplantation (median 180 d). All relapses occurred in patients with acute leukaemia less than or equal to 18 years of age with a high risk for relapse, i.e. transplanted in second or later remission or with more than 10% blasts in the marrow before transplantation. Among all patients the probability of relapse was increased in patients without cytomegalovirus (CMV) infection (P = 0.001) and in patients without chronic GVHD (P = 0.049). Among leukaemic patients less than or equal to 18 years of age with a high risk of relapse all relapses occurred in patients (n = 11) without CMV infection, whereas no relapses were seen in patients (n = 13) with CMV infection (P = 0.006). Known risk factors for leukaemic relapse were comparable in both groups.     

Changes of inducible protein-10 and regulated upon activation, normal T cell expressed and secreted protein in acute rejection of pancreas transplantation in rats

AIM: To investigate the role of IFN-γinducible protein -10 (IP-10) and regulated upon activation, normal T cell expressed and secreted (RANTES) protein in acute pancreatic allograft rejection in rats. METHODS: An experimental pancreas transplantation model was established using diabetic SD rats as the recipient, induced by applying streptozocin (STZ). Pancreas transplantation was performed with a physiologic method of portal venous and enteric drainage. Rats were divided into two groups, isograft group (group A, n = 24) and allograft group (group B, n = 24) in which either healthy SD rats or Wistar rats served as donors, respectively. Twelve diabetic or healthy SD rats were used as controls. At d 1, 4, 7, and 10 post transplantation, serum IP-10 and RANTES were assessed by ELISA and their expression in the allografts was determined by immunohistochemistry. RESULTS: In group B (allograft group), the development of acute rejection was significantly correlated with increased serum concentration and tissue expression of IP-10 and RANTES, with a peak level at d 7 post transplantation. In contrast, there was no obvious change before and after transplantation in group A (isograft group). CONCLUSION: Our study suggests a possible role of IP-10 and RANTES in acute rejection and early monitoring of chemokines may be helpful in predicting the outcome of pancreas transplantation.     

Familial peroxidase-deficiency and acute myeloic leukemia (author's transl)]

A complete lack of myeloperoxidase (MPO) was demonstrated in a boy suffering from acute myeloic leukemia during the acute phase of the disease and after a remission was achieved. A partial defect of MPO was demonstrated in the patient's father, no further abnormalities were seen in other members of the family. The fine structure of the patient's neutrophils and monocytes appeared normal, no activity of MPO was demonstrated on the fine structural level. In the father's neutrophils transitional forms between cells exhibiting a normal MPO activity and those without activity were demonstrated. The neutrophil bactericidal activity was strongly inhibited in the patient and decreased in his father. Normal values were found in: NBT test, chemotaxis, serum-dependent phagocytosis, number of B and T lymphocytes, serum immunoglobulins, and complement. A possible connection between MPO deficiency and leukemia is discussed.