Degradation of heparan sulfate in the subendothelial extracellular matrix by a readily released heparanase from human neutrophils. Possible role in invasion through basement membranes.

Freshly isolated human neutrophils were investigated for their ability to degrade heparan sulfate proteoglycans in the subendothelial extracellular matrix (ECM) produced by cultured corneal and vascular endothelial cells. The ECM was metabolically labeled with Na2(35S)O4 and labeled degradation products were analyzed by gel filtration over Sepharose 6B. More than 90% of the released radioactivity consisted of heparan sulfate fragments 5-6 times smaller than intact heparan sulfate side chains released from the ECM by either papain or alkaline borohydride. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of either heparin or serine protease inhibitors. In contrast, degradation of soluble high molecular weight heparan sulfate proteoglycan, which was first released from the ECM, was inhibited by heparin but there was no effect of protease inhibitors. These results indicate that interaction of human neutrophils with the subendothelial ECM is associated with degradation of its heparan sulfate by means of a specific, newly identified, heparanase activity and that this degradation is facilitated to a large extent by serine proteases. The neutrophil heparanase was readily and preferentially released (15-25% of the cellular content in 60 min) by simply incubating the cells at 4 degrees C in the absence of added stimuli. Under these conditions, less than 5% of the cellular content of lactate dehydrogenase, lysozyme, and globin degrading proteases was released. Further purification of the neutrophil heparanase was achieved by its binding to heparin-Sepharose and elution at 1 M NaCl. It is suggested that heparanase activity is involved in the early events of extravasation and diapedesis of neutrophils in response to a threshold signal from an extravascular inflamed organ.

Subcellular localization of heparanase in human neutrophils.

The subcellular localization of a heparan sulfate degrading endoglycosidase, heparanase, was studied in human neutrophils. Unstimulated cells were disrupted by nitrogen cavitation and fractionated on a Percoll density gradient into three components, separating the plasma membranes, specific granules, and azurophilic granules. Heparanase activity was measured by gel filtration analysis of 35S-labeled degradation fragments released from subendothelial extracellular matrix (ECM) or produced during incubation with soluble, ECM-derived, heparan sulfate proteoglycans. Heparanase activity was found mainly in fractions containing the specific granules; this activity was inhibited by heparin. Freezing and thawing was not needed for recovery of the enzyme from the subcellular fraction, confirming previous data about its ready release. The mechanism of the ready release of heparanase from the specific granules requires further investigation.

Acute lymphoblastic leukemia. Association with vasopressin-responsive diabetes insipidus.

A rare case of acute lymphoblastic leukemia presenting with vasopressin-responsive diabetes insipidus (DI) is reported. The patient presented with polydypsia and polyuria of 9 L/day. Findings from special investigations of the CNS, including brain scan, computerized tomographic scan, EEG, and lumbar puncture were within normal limits. The patient's condition improved substantially after receiving vasopressin injections and later chlorpropamide. The incidence and underlying mechanism of this rare complication of acute leukemia are reviewed, and the response of DI to chlorpropamide is discussed briefly. In this patient it is presumed that the DI was caused by leukemic infiltration of the supraopticohypophyseal tract, posterior pituitary, or hypothalamus.

Effect of theophylline on erythrocytosis in chronic obstructive pulmonary disease.

BACKGROUND: Patients with chronic obstructive pulmonary disease (COPD) tend to develop secondary erythrocytosis to compensate for their chronic hypoxia. Theophylline has recently been shown to reduce hematocrit and erythropoietin blood levels in normal subjects and in patients with erythrocytosis after renal transplantation. OBJECTIVE: To determine whether theophylline may be used to lower the hematocrit in patients with COPD. METHODS: Two hundred four patients with COPD were studied retrospectively and 10 patients prospectively (8 starting treatment with the drug [group 1] and 2 who suspended its long-term use [group 2]) for the correlation between theophylline therapy and hematocrit and erythropoietin level. RESULTS: In the patients studied retrospectively, lower hematocrits were found in the theophylline-treated than in the untreated patients (0.43 +/- 0.006 vs 0.46 +/- 0.007, respectively; P < .002). Twelve untreated patients and 2 of those treated with theophylline had hematocrits above 52%. Oxygen saturation levels were similar in both groups, and exclusion of patients with oxygen saturation lower than 88% did not change the pattern, suggesting that the effect of theophylline could not be entirely explained by improved oxygen availability. Seven of the 8 patients studied prospectively in group 1 (P < .02) and the 2 patients in group 2 showed inverse correlations between hematocrits and theophylline administration. A similar pattern was observed with serum erythropoietin levels in 5 of 7 patients studied. The effects were reproducible on rechallenge in 3 of the 4 patients in group 1 and the 2 patients in group 2. CONCLUSIONS: Theophylline may have a beneficial effect in treatment and prevention of erythrocytosis in patients with COPD.

Hypophosphatemia in a patient with lymphoma in leukemic phase.

A patient with histiocytic lymphoma had abdominal masses, hypophosphatemia, normocalcemia, and a normal serum parathyroid hormone value. After chemotherapy, transient hyperphosphatemia ensued, the abdominal masses resolved, and other manifestations of the disease were suppressed. One week after discontinuation of the chemotherapy, the abdominal masses and other signs indicative of reactivation of the malignant disease reappeared. During the relapse, the serum phosphorus level fell to 0.7 mg/dL, and urinary excretion of phosphorus became negligible. After resumption of chemotherapy, serum concentration and urinary excretion of phosphorus increased. These observation suggest that severe hypophosphatemia may be a complication of hematologic neoplasia. It is proposed that this abnormally may be caused by a shift of excessive amounts of extracellular phosphorus into the rapidly replicating malignant cells.

Proposed mechanism of the inflammatory attacks in familial Mediterranean fever.

Peritoneal and synovial fluids of patients with familial Mediterranean fever lack a protein that inhibits neutrophil chemotaxis by antagonizing the complement-derived inflammatory mediator C5a. The C5a inhibitor activity was studied with the use of a C5a binding assay where peritoneal fluids were tested for their ability to inhibit recombinant C5a binding to dibutyryl cyclic adenosine monophosphate-induced U937 cells. In contrast to normal peritoneal fluids, those from patients with familial Mediterranean fever contained less than 1% C5a inhibitor activity. Gel filtration and ion exchange chromatography of peritoneal fluids from those patients did not yield any fraction that inhibited C5a binding. We suggest that the serosal tissue of patients with familial Mediterranean fever is devoid of C5a inhibitor activity and that this deficiency may explain in part the local inflammatory episodes characteristic of this disease.

MEFV mutations in Turkish patients suffering from Familial Mediterranean Fever.

Familial Mediterranean fever (FMF) is a recessive inherited disorder affecting Sephardic Jews, Arabs, Armenians and Turks. The gene responsible for FMF was recently cloned and several disease-associated mutations have been described. We have evaluated seven MEFV mutations in 460 chromosomes of 230 unrelated patients with FMF living in Turkey, using PCR methods. The M694V allele accounted for 43.5% of the alleles studied and 19.1% of the patients were homozygous. The M680I, V726A and M694I mutations were responsible for 12.0%, 11.1% and 2.8% of the patients respectively. R761H, K695R and E148Q were rarely encountered. Two thirds of the disease alleles were attributed to three common mutations: M694V, M680V and V726A, but only 54% of the patients carried one or two of the three mutations. Adding the four rarer mutations increased these figures to 72% and 60%, respectively. Altogether, 79.6% of the patients bore at least one of the main mutations, and 84.3% carried at least one of the seven mutations studied. The 28 patients suffering also from amyloidosis carried at least one of five mutations, M694V being the most common. These results suggest that the origin of FMF in Turkey is heterogenous, all common mutations are associated with amyloidosis. Further, rapid and accurate molecular diagnosis of FMF is feasible in most cases.

Disorders of neutrophil function.

Major bacterial infections are most commonly associated with agranulocytosis or an abnormality of immunoglobulins or complement. Occasionally, repeated infections cannot be attributed to these relatively common causes. In such cases, a quantitative abnormality in neutrophil function should be sought. Complete evaluation of neutrophil function, including: chemotaxis, adhesion, aggregation, phagocytosis, granule content and degranulation, respiratory burst activity and bacterial killing is expensive and requires the services of a specialized laboratory. However, preliminary screening of a patient with a predisposition towards infection can be carried out using simple and inexpensive methods. These include examination of blood films, chemotaxis assay, NBT test and peroxidase staining. For final diagnosis and determination of genetic transmission and treatment, specific tests are indicated. Investigation of neutrophil functions may be useful for the diagnosis of congenital and acquired neutrophil disorders. These assays may also be useful in research, diagnosis and follow up of non-infectious diseases with active inflammatory component.

Specific binding of placental acidic isoferritin to cells of the T-cell line HD-MAR.

Acidic placental isoferritin inhibited the blastogenic response of peripheral human lymphocytes to T-cell activating lectins. We measured specific binding of radioiodinated placental isoferritin to cells of the T-cell line HD-MAR and found specific high-affinity binding. Binding was faster and more ferritin was bound at 37 degrees C than at 4 degrees C. Displacement experiments indicated that most of the binding occurred at the cell surface. Acidic placental ferritin and isolated H-type ferritin subunits but not isolated L-type subunits, competed for the binding. Scatchard plot analysis showed characteristics of a single binding species with a dissociation constant (Kd) of 1.3-4.4 x 10(-11) M. The results suggest the presence of receptors for acidic isoferritin on T-lymphocytes and thus, a regulatory role for the acidic ferritin H-type subunit in T-cell function.

HLA-B51 may serve as an immunogenetic marker for a subgroup of patients with Behçet's syndrome.

Epidemiologic data, family history, clinical data, HLA typing, neutrophilic chemotaxis, and immunofluorescence of clinically normal non-sun-exposed skin were studied in 46 Israeli non-Ashkenazi Jewish and Arab patients with Behçet's syndrome. HLA-B51 was present in 71 percent of the patient group as compared with 13 percent of the control group (relative risk = 17.1). In four of 30 families in the B51-positive group, there was a close relative of the proband with Behçet's syndrome who was carrying the HLA-B51 antigen. Neutrophilic chemotaxis in this group was enhanced in 80 percent of the patients, and in most patients no deposition of immunoglobulin in the dermo-epidermal junction was observed, whereas C3 was present in papillary vessels. In the B51-negative group, the family history was negative for Behçet's syndrome, neutrophilic chemotaxis was enhanced in only two of eight patients, and in four of six patients, IgM deposition was detected in the dermo-epidermal junction. It is concluded that in Israeli non-Ashkenazi Jews and Arabs, there is a significant association between HLA-B51 and the risk of developing Behçet's syndrome. The B51-positive patient group has a family history of the disease, enhanced neutrophilic chemotaxis, and a lack of immunoglobulin deposition in the dermo-epidermal junction.

Widespread expression of serum amyloid A in histologically normal human tissues. Predominant localization to the epithelium.

Serum amyloid A (SAA) is an acute-phase reactant whose level in the blood is elevated to 1000-fold as part of the body's responses to various injuries, including trauma, infection, inflammation, and neoplasia. As an acute-phase reactant, the liver has been considered to be the primary site of expression. However, limited extrahepatic SAA expression was described in mouse tissues and in cells of human atherosclerotic lesions. Here we describe nonradioactive in situ hybridization experiments revealing that the SAA mRNA is widely expressed in many histologically normal human tissues. Expression was localized predominantly to the epithelial components of a variety of tissues, including breast, stomach, small and large intestine, prostate, lung, pancreas, kidney, tonsil, thyroid, pituitary, placenta, skin epidermis, and brain neurons. Expression was also observed in lymphocytes, plasma cells, and endothelial cells. RT-PCR analysis of selected tissues revealed expression of the SAA1, SAA2, and SAA4 genes but not of SAA3, consistent with expression of these genes in the liver. Immunohistochemical staining revealed SAA protein expression that co-localized with SAA mRNA expression. These data indicate local production of the SAA proteins in histologically normal human extrahepatic tissues.

The inhibitory effect of heparin and related glycosaminoglycans on neutrophil chemotaxis.

Clinical observations have shown that heparin has antiinflammatory activities. The effect of heparin on neutrophil chemotaxis was evaluated in vitro in the Boyden Chamber. This method enabled differentiation between the direct effects of heparin on neutrophil migration and locomotion, and its effects on chemotactic factors. Heparin inhibited both the random migration and directed locomotion of human neutrophils toward zymosan-activated serum (ZAS) and F-met-leu-phe (FMLP). Inhibition was found to be dependent on the concentrations of the heparin and of the chemotactic factors. No specific binding of heparin to the neutrophils could be demonstrated, and heparin's inhibitory effects were eliminated by simple washing of the cells. When added directly to the chamber containing chemotactic factor, heparin inhibited the chemotactic activity of ZAS but not that of FMLP, suggesting a direct inhibitory effect against C5a, the principal chemotactic factor in ZAS. Experiments performed with low-molecular-weight heparin, N-desulfated heparin, dextran sulfate, chondroitin sulfate and dextran indicated that the inhibitory effects of heparin on neutrophil chemotaxis are not related to its anticoagulant activity, but probably depend on the degree of sulfation of the heparin molecule.

Partial resistance to anticoagulation after streptokinase treatment for acute myocardial infarction.

This study examines the response of 3 different groups of patients to anticoagulants: 50 patients previously treated with streptokinase for acute myocardial infarction (AMI) (group 1), 24 patients with AMI who had received anticoagulants without prior thrombolysis (group 2) and 11 subjects who received anticoagulants for noncoronary indications (group 3). No significant differences were detected between groups 2 and 3; therefore, they were combined for analysis. After streptokinase, patients required 37,755 +/- 1,516 (mean +/- standard error of the mean) U of heparin per day to achieve the desired activated partial thromboplastin time (APTT). The dosage was 30,294 +/- 1,089 U/day in patients without antecedent thrombolysis (p less than 0.001). Group 1 patients required 5 +/- 0.4 days until adequate anticoagulation was achieved, compared with 3 +/- 0.2 days in the control group (p = 0.01). Despite higher heparin requirements, group 1 patients had a lower APTT value than the other subjects (87 +/- 5 vs 101 +/- 6 seconds, p = 0.08). Group 1 patients required 5 +/- 0.3 days to reach anticoagulation with warfarin versus 4 +/- 0.2 days in groups 2 + 3 (p = 0.05). Comparison of groups 1 and 2 yielded similar, although smaller, differences. Patients treated with streptokinase for AMI seem to be partially resistant to anticoagulation, which may increase the risk of reocclusion.

Plasma cell leukemia and myeloma: a scanning electron-microscopic study of cell surface features in six cases.

Circulating plasma cells from six patients who had plasma cell leukemia were examined by transmission and scanning electron microscopy. In all cases, leukemic plasma cells constituted more than 60% of the total cell population in the peripheral blood. Transmission electron microscopy confirmed that the leukemic cells were plasmacytic and that many of them contained parallel arrays of rough endoplasmic reticulum and a prominent Golgi apparatus. Scanning electron microscopy confirmed previous observations of cultured myeloma cells and showed that plasma cells display varying numbers of surface blebs in addition to short stublike microvilli. The microvilli were frequently clustered together in one area of the surface. Bleb formation appears to be characteristic of plasma cells, but its nature is still obscure. Current knowledge of this phenomenon is briefly reviewed.

Neutrophil function studies in clinical medicine.

A complete evaluation of neutrophil function including: chemotaxis; adhesion; aggregation; phagocytosis; granule content and degranulation; respiratory burst activity; and bacterial killing; is expensive and requires the services of a specialized laboratory. However, preliminary screening of a patient with a predisposition toward infection, can be carried out using simple and inexpensive methods. These include examination of blood films, which may prove helpful in the diagnosis of Chediak-Higashi syndrome and specific granule deficiency; the Rebuck skin window test, which estimates chemotactic defects; the NBT test, which screens for chronic granulomatous disease patients; and peroxidase staining of the blood film in order to estimate the content of myeloperoxidase, when myeloperoxidase deficiency is suspected. For final diagnosis and determination of genetic transmission and radical treatment, ie, bone marrow transplantation, specific tests are indicated. Neutrophil function studies have also proved useful in detecting diseases in which defects in neutrophil function are secondary to the primary disorder. Indeed, increased neutrophil chemotaxis has been reported in the active phase of diseases such as: familial Mediterranean fever; psoriasis vulgaris, Behcet's syndrome and Sweet's syndrome. In these disorders the neutrophil chemotaxis assay has aided in the diagnosis and follow-up, particularly in evaluating the response to antiinflammatory agents, such as colchicine.

Therapy-related myelodysplastic syndrome. Two cytogenetically unrelated abnormal clones in a patient with multiple myeloma.

A multiple myeloma patient presented for cytogenetic analysis at diagnosis of secondary MDS, which followed cytotoxic treatment including melphalan. Two abnormal unrelated clones were detected, one of them had 5q-, 7q- with clonal evolution of an additional aberration, t(12;13); in the second clone there was a translocation between the two homologues of chromosome 1 as the only aberration. We suggest that the clone with 5q- and 7q- represented the secondary MDS cells, whereas the abnormal clone with t(1;1) represented the plasmablasts of the multiple myeloma.

Neutrophil Function in Hematological Neoplasia.

Though the main reason for infection in hematologic malignancies remains the reduction in the neutrophil count, there are certain conditions where agranulocytosis alone could not account for this tendency. In non lymphatic disorders, obvious modifications in mature neutrophil functions have been described. They predict susceptibility to infection and bad prognosis in myelodysplastic syndromes (MDS) and in acute non lymphatic leukemia (ANLL) where the mature looking neutrophils represent the leukemic clone and function aberrantly. The defects in neutrophil function encountered in myeloproliferative diseases including chronic myeloid leukemia, are mostly of scientific interest, and usually do not alter the clinical course of the disease in these patients. The alterations in neutrophil function in the lymphoproliferative disorders result mostly from humoral defects, mainly immunoglobulin deficiency and a variety of neutrophil inhibitory substances, and are not due to primary cellular defects. Neutrophil dysfunction may also result from a protracted infectious course and various treatment modalities, includng splenectomy and chemo-radiotherapy. Neutrophil function studies in splenectomised patients and in selected cases with MDS and ANLL, and studies of serum immunoglobulins and neutrophil inhibitors in patients with lymphoid diseases, may be useful in screening those cases who are prone to infectious problems, and who might therefore benefit from increased infectious precautions.

Glycosylated serum ferritin in patients with hematological malignancies before and after bone marrow transplantation.

Glycosylated and total serum ferritin levels were monitored in patients with acute leukemia and lymphoma undergoing bone marrow transplantation (BMT). Serum ferritin was high in relapsing patients and normal in most patients in complete remission (CR). In patients with an uncomplicated course, levels of ferritin increased during the first month after BMT with subsequent decrease. Three patients with lymphoma and five with acute leukemia had high serum ferritin levels despite achieving apparent complete hematological remission which was of short duration. The results were compared with groups of lymphoma patients at presentation and during remission and with healthy normal controls. In all the lymphoma patients and in 3 of the 5 leukemia patients the percent of ferritin glycosylation was normal at CR. It was low at the time of diagnosis in all patients. Thus, the percent glycosylation proved a more reliable marker for clinical remission than total serum ferritin. During follow up after BMT in uncomplicated cases, the percent of glycosylated ferritin returned to normal levels earlier than the total serum ferritin. These findings indicate that the evaluation of the amount of glycosylated ferritin may provide useful information in hematological patients in whom there is a discrepancy between high serum ferritin levels and the clinical condition.

Molecular diagnosis of FMF: lessons from a study of 446 unrelated individuals.

BACKGROUND: Traditionally, the diagnosis of familial Mediterranean fever (FMF) has been based on clinical manifestations and the physician's experience. Following the cloning of the gene associated with this disease (MEFV), genetic analysis of its mutations has become available, providing a new tool for the establishment or confirmation of the diagnosis of FMF. OBJECTIVES: We analyzed the results of molecular testing for MEFV mutations in 600 individuals. We wished to determine how many of them bore mutations and what percentage had clinically active FMF. We also compared the rate of genetic confirmation of the FMF diagnosis in referrals with suspected FMF seen by general practitioners with that of persons sent for genetic analysis by FMF experts. METHODS: Of 600 individuals tested for FMF mutations, we analyzed separately 446 unrelated persons for the combination of their mutations, epidemiological data, and clinical manifestations. The five most common mutations in the present cohort were analyzed using the amplification refractory mutation system (ARMS). RESULTS: Of the 446 subjects analyzed, 249 (55%) bore mutations: 147 of these were homozygotes or compound heterozygotes, all of whom had FMF according to clinical criteria. Of the remaining 102 heterozygotes, 72 had FMF according to clinical criteria. Two patients with none of the five mutations also had FMF: North African Jews bore mainly mutations M694V and E148Q. The M6941 mutation was found exclusively in Palestinian Arabs. The rate of confirmation of FMF diagnosis by mutation analysis in subjects sent by FMF experts was significantly higher than that of persons referred by general practitioners. Analysis of the molecular testing of the multicase families (154 individuals) revealed that 141 of them bore MEFV mutations and that 4 persons homozygous for E148Q were asymptomatic. CONCLUSIONS: Molecular analysis of FMF mutations confirmed the diagnosis in about 60% of the referrals with suspected FMF. Some (33%) of the patients were heterozygotes, and there were also FMF patients with none of the 5 mutations analyzed. A second opinion by an FMF expert may decrease the need for mutation analysis in subjects suspected of having FMF.

Inhibition of neutrophil activation by fibrinogen.

Physiological levels of human fibrinogen markedly inhibited the chemotactic activity of human neutrophils triggered by zymosan-activated serum (ZAS), C5a, or IL-8 in a Boyden chamber assay. Fibrinogen also slightly inhibited the N-formyl-methionyl leucyl-phenylalanine (FMLP)-induced migration of human neutrophils. Albumin was devoid of the inhibitory activities displayed by fibrinogen in this system. The inhibition of chemotaxis by fibrinogen was dose-dependent and saturable. Fibrinogen placed in the upper compartment of the Boyden chamber produced a larger inhibition than that obtained with fibrinogen placed in the lower compartment. Lysine as well as the lysine analog 6-aminohexanoic acid (AHA) decreased the inhibitory capacity of fibrinogen. In contrast, both arginine and glutamine failed to suppress the fibrinogen-mediated inhibition of neutrophil chemotaxis. AHA counteracts the inhibition of ZAS-induced chemotaxis by anti-CD18 monoclonal antibody, suggesting that lysine binding sites are required for integrin function in chemotaxis. Fibrinogen also inhibited, in a dose-dependent manner, the oxygen consumption of neutrophils activated by opsonized zymosan. Taken together, the present results indicate that fibrinogen modulates neutrophil functions and suggest that in addition to its role in blood coagulation, circulating fibrinogen may be involved in regulation of the inflammatory response.