Precise quantification of haemoglobin in erythroid precursors and plasma.

INTRODUCTION: Haemoglobin (Hb) quantification in whole blood is possible by various spectrophotometric methods. However, determination of low-level Hb in erythroid precursors or haemolytic plasma is inaccurate because of contribution from light scatter and/or nonhaemoglobin components with overlapping absorbance. Therefore, this study developed a sole method allowing accurate spectrophotometric quantification of Hb at a low concentration range. METHODS: Advantage was taken of the unique absorption spectra of carbon monoxide-Hb complex (COHb) as compared to oxyHb. The visible absorption spectra of samples were recorded prior and following carbon monoxide exposure. A difference extinction coefficient at the maximal difference absorption was used to calculate Hb concentrations. RESULTS: Known amounts of Hb were added to mouse erythroleukaemia (MEL) cells lysate or plasma to yield known 'theoretical' concentrations. The concentrations were measured by the current and known methods. The current method was found much more accurate compared with previous methods specifically at low concentrations. CONCLUSION: The method is valid for accurate quantification of Hb at a wide concentration range (>0.1 µm/L) in erythroid precursors or plasma and is optional for other biological fluids.

Pivanex, a histone deacetylase inhibitor, induces changes in BCR-ABL expression and when combined with STI571, acts synergistically in a chronic myelocytic leukemia cell line.

Chronic myelogenous leukemia (CML) is associated with the high TK activity chimeric protein BCR-ABL, known to contribute to cell tumorogenicity, resistance to apoptosis and differentiation. STI571, the TK inhibitor, is the current treatment for CML. One possible approach to overcome STI571 resistance appearing in some cases, involves the combination of histone deacetylase inhibitors (HDI) and STI571. We demonstrated that in K562, the CML cell line, pivaloyloxymethyl butyrate (Pivanex)-induced apoptosis, differentiation and reduced BCR-ABL protein levels and that the combination of Pivanex with STI571 acted synergistically. These data suggest the possible benefit of combining this HDI with STI571 for treatment of CML.

Vascular damage by unstable hemoglobins: the role of heme-depleted globin.

The study compared the damage inflicted to endothelial cells (ECs) by intact hemoglobin (Hb) and isolated chains. To compare optional in vivo contact of acellular Hb with the endothelium, oxy-forms of Hb and its isolated alpha- and beta-chains existing in the thalassemias were added to primary confluent cultures of bovine aorta EC. Cell damage was followed by morphological changes or leakage of lactic dehydrogenase and pre-inserted 51Cr from the cells, followed for 27 h. Under these experimental conditions, Hb did not affect the cells but its chains inflicted damage, beta- more than alpha-chains. Based on the literature and our data, we hypothesized that hemin and/or globin should be responsible for the increased endothelial damage by beta-chains. While hemin hardly affected ECs, globin, unlike the plasma protein hemopexin, was harmful. Since hemin release leaves globin with a large hydrophobic surface, the globin-damage appears to result from adsorptive pinocytosis to endothelial membrane.

Serum CA 125 levels in patients with chronic lymphocytic leukemia.

Cancer antigen 125 (CA 125) is a glycoprotein expressed in normal tissues originally derived from celomic epithelia and its serum level is elevated in various benign and malignant conditions that involve stimulation of these tissues. Elevated levels have been reported in 40-43% of patients with non-Hodgkin's lymphoma (NHL) at diagnosis and were associated with parameters known to be associated with advanced and disseminated disease, and with event-free and overall survival. No difference in CA 125 level was found between indolent and aggressive lymphomas, and four of six patients with small lymphocytic lymphoma had elevated CA 125 levels. We therefore decided to measure serum CA 125 levels in chronic lymphocytic leukemia (CLL) patients, and evaluated them in 74 consecutive patients. The mean time from diagnosis to test was 74.5 months (range: 0-300). The mean serum CA 125 level was 16.3 U/ml (range: 3.7-133, normal value: <35 U/ml). CA 125 levels were elevated only in two patients (2.7%). To conclude, serum CA 125 levels are rarely elevated in CLL patients. It is possible that serum CA 125 levels can help differentiate between equivocal cases of small lymphocytic lymphoma and CLL.

All three receptors for vascular endothelial growth factor (VEGF) are expressed on B-chronic lymphocytic leukemia (CLL) cells.

B-chronic lymphocytic leukemia (B-CLL) cells have a long survival owing to an alteration in the normal pathways of apoptosis. CLL cells have been found to produce and secrete vascular endothelial growth factor (VEGF). In addition to its major role in angiogenesis, VEGF affects cell survival by interfering with apoptosis. The aim of the present study was to investigate the expression of the VEGF receptors VEGFR-1, VEGFR-2, and VEGFR-3 on B-CLL cells, singly and combined. B-CLL cells were isolated from peripheral blood drawn from patients with CLL. Total VEGF receptor, examined in 13 samples by flow cytometry was present in all cases with mean CD19+/VEGF+ expression of 76% (range 52-92%). Specific receptor expression, examined in 27 samples by immunocytochemical methods, was positive for VEGFR-1 in all 27 patients and for VEGFR-2 and VEGFR-3 in 26 (96%). These findings suggest that the VEGF transduction pathway may be very active in CLL cells, and both its paracrine and autocrine pathways may contribute to their enhanced survival.

Expression of basic fibroblast growth factor is associated with poor outcome in non-Hodgkin's lymphoma.

It is now clear that angiogenesis and angiogenesis factors are important in the pathogenesis of haematological malignancies. High pretreatment levels of serum basic fibroblast growth factor have been shown to be associated with poor prognosis in patients with non-Hodgkin's lymphoma. The aim of this study was to evaluate whether non-Hodgkin's lymphoma cells express basic fibroblast growth factor and/or its receptor (fibroblast growth factor receptor-1) and whether basic fibroblast growth factor expression correlates with basic fibroblast growth factor serum levels, intratumoral microvessel density, and patient outcome. We measured basic fibroblast growth factor by enzyme-linked immunosorbent assay in sera taken from 58 patients with non-Hodgkin's lymphoma before treatment and in 19 of them also after treatment. Pathological specimens at diagnosis were evaluated by immunohistochemistry staining using polyoclonal antibody against factor-VIII-related antigen, basic fibroblast growth factor and fibroblast growth factor receptor-1 to determine the expression of the microvessel count and basic fibroblast growth factor and fibroblast growth factor receptor-1. The lymphoma specimens demonstrated positive staining for basic fibroblast growth factor (in 23%) and fibroblast growth factor receptor-1 (in 58.5%). The patients who expressed basic fibroblast growth factor had a significantly worse progression-free and overall survival than those who did not (P=0.003 and P=0.03 respectively), while patients expressing fibroblast growth factor receptor-1 were less likely to achieve complete remission than those lacking the receptor (33% vs 65%, P=0.047). There was no correlation of basic fibroblast growth factor staining with either serum basic fibroblast growth factor levels or microvessel count. Basic fibroblast growth factor serum levels did not change significantly after treatment These results suggest that non-Hodgkin's lymphoma specimens express basic fibroblast growth factor and its receptor (fibroblast growth factor receptor-1) and this expression is associated with poor patient outcome.

Bcl-2 expression correlates positively with serum basic fibroblast growth factor (bFGF) and negatively with cellular vascular endothelial growth factor (VEGF) in patients with chronic lymphocytic leukaemia.

A large proportion of B-chronic lymphocytic leukaemia (B-CLL) cells express the anti-apoptotic protein Bcl-2. Basic fibroblast growth factor (bFGF) has been shown to upregulate the expression of Bcl-2 in B-CLL cell lines. Vascular endothelial growth factor (VEGF) has been shown to enhance the survival of endothelial cells by upregulating the expression of Bcl-2. In the present study, we measured serum and cellular levels of bFGF and VEGF in 85 patients with CLL using a commercial quantitative sandwich enzyme immunoassay technique. Levels of Bcl-2 were also assayed concomitantly using Western blot analysis. The mean serum level of bFGF was 53.4 pg/ml (range 0-589) and that of VEGF 459.2 pg/ml (range 33-1793). The mean cellular level of bFGF was 158.3 pg/2 x 105 cells (range 0.8-841) and VEGF, 42.4 pg/2 x 105 cells (range 0-244). A high correlation was found between serum and cellular bFGF levels (P < 0.001), but not between the corresponding VEGF levels. Twenty-nine of 69 patients (42%) evaluated for Bcl-2 level, expressed it. The Bcl-2 level was positively correlated with the serum bFGF level (P = 0.007). However, surprisingly there was a negative correlation between Bcl-2 expression and intracellular VEGF level (P = 0.003). A positive correlation was also found between serum bFGF and disease follow-up time and log white blood cell count. These findings indicate that in CLL there is a correlation between angiogenesis-related factors and apoptosis-related protein expression, and elevated bFGF levels may account for the elevated Bcl-2 levels.

Doxorubicin and a butyric acid derivative effectively reduce levels of BCL-2 protein in the cells of chronic lymphocytic leukemia patient.

B-chronic lymphocytic leukemia (B-CLL) is a disease caused primarily by defects in the apoptosis mechanism. AN-9, a butyric acid (BA) derivative, is a potent differentiating and an anti-cancer drug that induces apoptosis in HL-60 cells. Herein we show the affect of AN-9, alone and in combination with doxorubicin, on cell cultures from B-CLL patients. Cells from 17 patients were cultured and tested for viability, apoptosis, bcl-2 and bax protein expression. Exposure of B-CLL cell cultures to AN-9 was accompanied by apoptosis and a marked viability loss (up to 46%, p=0.0017). AN-9 reduced up to 51% (p=0.0017) the levels of bcl-2 in 57% of the cultures that express bcl-2. The combination of low concentrations of AN-9 and doxorubicin more than additively enhanced apoptosis and reduced bcl-2 levels in B-CLL cultures which were resistant to AN-9. AN-9 enhanced bax expression up to 58%(p=0.008) in cultures from 53% of the patients, but had no effect on bax levels when combined with doxorubicin. In conclusion, AN-9 alone reduced bcl-2 and enhanced bax expression in cultures from B-CLL patients, and the reduction of bcl-2 levels in combination with doxorubicin was greater than additive. These results may be beneficial in possible future combination therapy with AN-9 in B-CLL.

Microvessel density in chemosensitive and chemoresistant diffuse large B-cell lymphomas.

Preliminary reports involving a number of different kinds of tumors have indicated that microvessel quantification may be useful in predicting disease outcome. The aim of this study was to examine the relationship between microvessel density (MVD) as a parameter of tumor angiogenesis and the response to chemotherapy in diffuse large B-cell (DLBC) lymphomas. A total of 36 DLBC lymphoma patients were evaluated, 23 of them with a chemosensitive; responsive disease (median survival 8y) and 13 with a chemoresistant, refractory disease (median survival 8 months). Microvessel quantification was performed by immunohistochemical staining, using monoclonal antibodies against factor VIII related antigen (F8RA) and against platelet/endothelial cell adhesion molecule-CD31. We found that F8RA stained a significantly higher number of blood vessels (about 2.5 times more) than CD-31; 7 samples were not stained with CD-31 but were positive for F8RA. There was no significant difference between the density of microvessel staining of the two groups. In the chemosensitive DLBC lymphomas positive for F8RA, the mean number of microvessels stained was 54.5 +/- 36.1 per microscopic field (200x) examined (range 6-149) whereas in the chemoresistant group the corresponding mean number was 43.1 +/- 25.5 (range 11-94). F8RA appears to be more sensitive for staining DLBC lymphomas microvessels than CD-31. Our data demonstrate that there is no correlation between tumor MVD and response to chemotherapy in patients with DLBC lymphomas.

Expression of bcl-2 and bax in cells isolated from B-chronic lymphocytic leukemia patients at different stages of the disease.

B-Chronic lymphocytic leukemia (B-CLL) is an example of a human malignancy caused by alterations in the pathways of programmed cell death. In this disease the anti-apoptotic protein, bcl-2, is overexpressed and may lead to the prolonged survival of a malignant CLL clone. In the present study we examined the expression of bcl-2 and bax, which has an antagonistic role against the function of bcl-2, in cells from CLL patients at different stages of the disease, by immunoblot analysis. A direct association between the stage of the disease and the level of bcl-2 in the patients' cells was observed. At stages A and B, 33% and 29% of patients, respectively, expressed high levels of bcl-2, as opposed to 80% of patients at stage C (p= 0.019). Bax level was not significantly associated with the stage of the disease, although patients at stage C had higher levels of bax. In this study we found a trend of association between bcl-2 and bax levels. Analysis including all patients revealed that 65% of the patients who expressed high levels of bcl-2 had high levels of bax (p = 0.058). Of patients in stage C, 45% expressed high levels of both bcl-2 and bax while 50% and 42% of patients at stages A and B had low levels of both proteins.

Protein disulfide isomerase mediates integrin-dependent adhesion.

Cell adhesion is mediated by the integrin adhesion receptors. Receptor-ligand interaction involves conformational changes in the receptor, but the underlying mechanism remains unclear. Our earlier work implied a role for sulfhydryls in integrin response to ligand binding in the intact blood platelet. We now show that non-penetrating blockers of free sulfhydryls inhibit beta(1) and beta(3) integrin-mediated platelet adhesion regardless of the affinity state of the integrin. Removal of the inhibitors prior to adhesion fully restores adhesion despite the irreversible nature of inhibitor-thiol interaction, indicating sulfhydryl exposure in response to adhesion. We further show that blocking protein disulfide isomerase (PDI) inhibits adhesion. These data indicate that: (a) ecto-sulfhydryls are necessary for integrin-mediated platelet adhesion; (b) disulfide exchange takes place during this process; (c) surface PDI is involved in integrin-mediated adhesion.

Pernicious anemia, gastric carcinoid, and autoimmune thrombocytopenia in a young woman.

The association between gastric carcinoid tumors and pernicious anemia is well recognized. Such tumors occur in the presence of achlorhydria, chronic atrophic gastritis, hypergastrinemia, and enterochromaffin-like cell hyperplasia. In this case report, a 29-year-old woman with pernicious anemia and autoimmune thrombocytopenia who developed gastric carcinoid tumors of the gastric body is described. This is the second description of pernicious anemia associated with autoimmune thrombocytopenia. This association in a young woman together with the therapeutic options and decisions that were taken in the treatment of the patient are discussed.

Uptake of pivaloyloxymethyl butyrate into leukemic cells and its intracellular esterase-catalyzed hydrolysis.

UNLABELLED: Pivaloyloxymethyl butyrate (AN-9), a butyric acid (BA) prodrug, exhibited low toxicity and significant anticancer activity in vitro and in vivo. The purpose of this study was to elucidate the basis for AN-9 increased anticancer activity compared to BA, by studying the uptake of BA and AN-9 into the cells. METHODS: The uptake rate and level of [14C]-AN-9 and [14C]-BA, labeled on the carboxylic moiety of BA, into HL-60 and MEL leukemic cell lines was measured. The cells were filtered and the retained radioactivity was determined. The dependence of the uptake on the activity of cellular esterases and membrane fluidity was investigated. RESULTS: The uptake level in cells incubated with [14C]-AN-9 increased rapidly, peaked after 30 min in MEL and 1 h in HL-60 cells, and declined thereafter. This decline could be attributed to the hydrolysis of AN-9 by cellular esterases and catabolism of the released BA to CO2. In cells pretreated with an esterase inhibitor and incubated with [14C]-AN-9, the reduction of radioactivity was less precipitous. In cells exposed to [14C]-BA, the intracellular radioactivity level was low and unaffected by treatment with an esterase inhibitor. The uptake of [14C]-AN-9 decreased significantly at 4 degrees C compared to that at 37 degrees C. CONCLUSION: The higher potency of AN-9 compared to BA could be at least partially attributed to the more rapid uptake of the lipophilic AN-9 and the release of BA in the cells.

Bcl-2, Bcl-X, Bax, and Bak expression in short- and long-lived patients with diffuse large B-cell lymphomas.

Long-term cure is now possible for approximately 50% of all patients with aggressive non-Hodgkin's lymphoma (NHL). Apoptosis-related proteins play an important role in the chemosensitivity or chemoresistance of tumors. We examined the role of Bcl-2 family proteins in aggressive NHL. We retrospectively selected two groups of patients by clinical outcome: 24 patients with chemoresponsive disease and long survival (median, 88 months); and 20 patients with chemoresistant disease and short survival (median, 8 months). The expression of the apoptosis-regulating proteins, Bcl-2, Bcl-X, Bax, and Bak, in the initial biopsy samples was examined with immunohistochemical methods. Specimens containing >10% immunostained tumor cells were considered immunopositive. An inverse association was found between length of patient survival and expression of Bcl-2, Bcl-X, and Bax. Bcl-2 was expressed in 75% of short-lived patients but in only 42% of the long-lived ones (P = 0.026). Bcl-X expression was also higher in the short-lived patients (40% versus 12.5%; P = 0.036). Unexpectedly, Bax expression was strongly associated with short survival (60% versus 21%; P = 0.008). Several combinations of protein expression, i.e., Bcl-2 with Bax, Bcl-2 with Bcl-X, and Bcl-X with Bax, were different between the groups: a positive expression of these proteins was found in the short-lived patients. Furthermore, a strong association was found between the expression of Bcl-2 and Bcl-X, suggesting that Bcl-X potentiates rather than replaces the effect of Bcl-2 in NHL. In diffuse large B-cell NHL, Bcl-2, Bcl-X, and Bax expression alone or in combination is associated with chemoresistance and shortterm survival.

Impaired T-lymphocyte function in B-chronic lymphocytic leukaemia as measured by the local xenogeneic graft-versus-host reaction.

The immunological dysfunction observed in B-chronic lymphocytic leukaemia (B-CLL) is often related to T-lymphocyte incompetence. The local xenogeneic graft-vs.-host reaction (XGVHR), an assay to evaluate T-lymphocyte function, was performed in 112 untreated B-CLL patients. The XGVHR results significantly correlated with clinical parameters: 37.1% of the patients in the stable phase (Rai stage 0-1-2) and only 13.3% of the patients in the progressive phase (Rai stage 3-4) had positive XGVHR results. Patients with negative results had a higher number and percentage of lymphocytes (25,247 vs. 17,071/microliter and 75.9% vs. 65.6%, respectively), much lower T/B lymphocyte ratio (0.37 vs. 0.93), higher WBC count (30,977 vs. 23,458/microliter), lower platelet count (158,068 vs. 181,684/microliter) and lower levels of IgA and IgM (115.6 vs. 200.5 mg/dl and 80.4 vs. 124.3 mg/dl, respectively) compared to those with positive results. Among those with negative XGVHR results, a higher mortality rate was found in those who had infections compared with those who did not (73.7% vs. 9.1%). In conclusion, the XGVHR assay significantly correlates with important characteristics of B-CLL and may be useful in the clinical evaluation of B-CLL patients.

Significance of BCR-ABL transcripts in bone marrow aspirates of Philadelphia-negative essential thrombocythemia patients.

Philadelphia-negative (Ph-neg) essential thrombocythemia (ET), polycythemia vera (PV) and idiopathic myelofibrosis (IMF) form a syndrome of related chronic myeloproliferative disorders (MPD) characterized by expansion of one or more of the hematopoietic progenitors. Based on our previous finding of BCR-ABL transcripts in bone marrow aspirates of 12/25 Ph-neg ET patients, we have expanded our study up to 40 patients. Here we describe the rational for performing this study and report 19 of 40 patients who have BCR-ABL transcripts in their BM, 11 of them carry b3a2 and 8 carry b2a2. The two groups, BCR-ABL positive and negative, were completely identical with regard to clinical characteristics and laboratory data. We also report preliminary results of our attempt to examine concordance or discordance of BCR-ABL expression in the peripheral blood and bone marrow of Ph-neg ET patients.

Transmission of HTLV-I to rats via peripheral blood mononuclear cells and serum from a patient with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) with amyotrophic lateral sclerosis (ALS)-like features.

We tested the possibility that lymphocytes and serum obtained directly from a patient with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) induce infection in rats. Inbred Fischer F344 immunosuppressed rats were inoculated intravenously with 10x10(6) peripheral blood mononuclear cells (PBMC; 3 rats) and serum (3 rats) obtained from a HAM/TSP patient, who was seropositive and polymerase chain reaction (PCR)-positive for the HTLV-I proviral genome. Antibodies to HTLV-I appeared in the rat sera 2 months later; rat peripheral blood lymphocytes, spleen, salivary gland, and spinal cord were found to contain the proviral genome. Control rats inoculated with normal donor PBMC and serum tested negative for the HTLV-I antibodies and for the HTLV-I proviral genome by PCR. The positive control F344 rats inoculated with 5x10(6) cells of a SLB-1 HTLV-I cell line were found to be infected after 2 months. This study demonstrates for the first time that HTLV-I can be transmitted not only by human cellular components but also by human cell-free sera in a rat model.

Red blood cell zinc protoporphyrin measurement for assessment of peripartum iron deficiency.

OBJECTIVE: To study the effectiveness of the rapid red blood cell zinc protorphyrin (RBC-ZPP) test for the detection of women with iron-deficiency anemia in the peripartum period. DESIGN: Blood was drawn prospectively from 150 healthy parturient women upon admission to the labor and delivery room and 72 hours after delivery. Concentration of RBC-ZPP was measured and correlated with hemoglobin level (p = 0.001), mean corpuscular volume (p = 0.002), hematocrit (p = 0.0001), platelet count (p = 0.002), and serum iron (p = 0.0001), serum ferritin (p = 0.0001) and serum transferrin (p = 0.0001) concentrations. RESULTS: RBC-ZPP concentration showed a significant increase from pre-delivery to 72 hours post-delivery. This change correlated significantly with the changes in all the other parameters studied. CONCLUSION: The RBC-ZPP test is a reliable, rapid, easy-to-perform, and inexpensive method of screening low-risk women, after uneventful vaginal delivery, for iron deficiency.

Ethnic cluster of HTLV-I infection in Israel among the Mashhadi Jewish population.

A high prevalence of human T-lymphotropic virus type I (HTLV-I) infection among Israeli Jews was previously reported. In the present study, screening for HTLV-I of Israeli Jews was expanded to 10 ethnic groups. HTLV-I antibodies were tested by the particle agglutination assay, ELISA, and by Western blot as a confirmatory method. The HTLV-I proviral genome was tested by nested PCR with tax primers (SK43/SK44 and Tr101/Tr102). The PCR tests were carried out in all seropositive subjects and the seronegative family members of the seropositives subjects in the Iranian population. Sixty-eight of the 1,679 subjects (4.1%) were found to be seropositive. The Jews originating from Mashhad had the highest infection rate of 60/306 (20%). Of the 479 Iranian non-Mashhadi Jews, 6 (1.3%) were seropositive. Of the 894 non-Iranian Israelis, only 2 (0.2%) were seropositive. HTLV-I proviral DNA was found in the peripheral blood lymphocytes of 66 out of 68 seropositive subjects and 6 out of 75 seronegative subjects. Sixty out of 123 (49%) Mashhadi Jews and 8 out of 14 (57%) non-Mashhadi Iranian Jews were PCR-positive. Three out of three seropositive non-Iranian Israelis were PCR positive. One non-Iranian Israeli (who originated from Ukraine) without family connections to the Iranian Jews was also PCR-positive. One hundred eighteen saliva samples (84 from subjects of Mashhadi origin, 31 from Iranian origin, and 4 of other origins) were also screened. Antibodies for HTLV-I were found in 23 out of 46 saliva samples from the individuals with particle agglutination (PA) and/or PCR-positive findings in blood. Twenty out of 23 PA-positive saliva samples also contained the proviral DNA. It is concluded that HTLV-I infection in Israel is mainly limited to Jews originating from Iran (most of them from Mashhad) and their family members.

HTLV-1 in mouthwash cells from a TSP/HAM patient and asymptomatic carriers.

Using in situ hybridization, the presence of T-cell lymphotrophic virus type I (HTLV-I) was shown in blood lymphocytes of one tropical spastic paraparesis (TSP/HAM) patient and in two asymptomatic carriers. HTLV-I was also detected in epithelial cells derived from mouthwash of the TSP/HAM patient. Mouthwash of one of the carriers showed an infected lymphocyte while mouthwash of the other carrier was negative. The infected epithelial cells stained both in the nucleus and in the cytoplasm, which indicated the presence of the virus in both subcellular compartments. Our observations suggest that saliva cells, lymphocytes and epithelial cells, may potentially participate in oral transmission of HTLV-I.