Deficiency of beta-glucuronidase in neutrophils from patients with precancerous states of the larynx.

Activity of beta-glucuronidase (GR) and acid phosphatase (AP) has been determined in peripheral blood neutrophils from 24 men with precancerous states of the larynx that is leukoplakia papillomas and pachydermia by means cytochemical methods described by Hayashi et al., and Barka and Anderson, respectively. The results obtained were expressed in terms of absolute counts of enzyme-positive and enzyme-negative cells with regard to enzyme activity variation within the enzyme-positive neutrophil population; the enzyme activity index score has been calculated. The control group consisted of 20 healthy subjects of the same sex. No significant alterations were found so far as AP activity is concerned between the group studied. In contrast, activity of GR in patients with precancerous states exhibited significant lowering. The most striking feature was in almost complete absence from the blood of GR-positive neutrophils with high activity of the enzyme. Majority of these cells showed only traces of the GR activity. According to authors opinion the deficiency of GR in neutrophils of patients with precancerous lesions pertains to problem of neutrophil-mediated cytotoxic effect against mammalian tumour cells.     

Intracellular enzymatic response of lymphocytes and neutrophils in patients with cancer of the larynx.

In 20 men, aged 35 to 55 years, with untreated cancer of the larynx activity of lysosomal acid phosphatase (AP), beta-glucuronidase (GR) and N-acetyl-beta-glucosaminidase was determined cytochemically in peripheral blood lymphocytes and neutrophils by means of Barka and Anderson, Hayashi et al. and Hayashi's method, respectively; the results obtained were compared with those in 20 healthy men aged 20 to 30 years. Total count of GR-positive lymphocytes was higher in the patients than in normal persons. Total counts of AP-, GR-, and GS-positive lymphocytes with not disrupted enzyme-positive lysosomal granules within the cell cytoplasm were significantly lower and total counts of cells exhibiting the disruption of lysosomal granules and the diffuse type of cytochemical reaction were significantly higher in the patients when compared with the control group. The response of neutrophils consisted of a significant elevation in numbers of AP-, and GS-positive cells; overall score of enzyme activity studied in neutrophils was not altered in the patients. The authors disucss the significance of their observations in the light of data on participation of lymphocytic and neutrophilic lysosomal apparatus in the immunological response against tumour specific antigen in patients with cancer.     

Leucine aminopeptidase of neutrophils and lymphocytes in patients with cancer

The leucine aminopeptidase activity has been determined by using the cytochemical method of Burston and Folk in peripheral blood neutrophils and lymphocytes of 45 patients with various malignancies. Lung cancer, carcinoma of the stomach and cancer of the colon was diagnosed in 24, 16, and 5 patients, respectively. Patients with metastases showed a significantly higher activity of the enzyme if compared with that in the control group of healthy subjects and patients without metastases. The percentage of enzyme-positive lymphocytes was elevated significantly in patients with metastases whereas a total percentage of lymphocytes with regard to differential leukocyte count was diminished both in patients with and without metastases. The absolute count of neutrophils was elevated both in patients with and without metastases. The authors discuss the significance of their observation with regard to the antitumor cytotoxic effect of neutrophils and lymphocytes.     

The activity of neutrophil myeloperoxidase in patients with lung carcinoma

MPO activity in the neutrophils has been evaluated in 25 patients with lung carcinoma. Surprisingly for us we observed no differences between patients and controls in the total MPO activity. However, we noted the increase in the percentage granulocytes with the first and fourth degree of scores in cancer patients in comparison to normal adults. The highest activity was characterized by the distribution of this enzyme not only in granules but also outside them. Any changes in the total activity of MPO may reflect the balance between the factors which may stimulate it as tumor promoters enhancing the production of superoxide anion or the inhibitory effect of plasma. Diffuse enzymatic reaction may be due to some functional abnormalities of phagocytosis or membrane damage by the MPO system. The role of MPO in anticancer mechanisms is briefly discussed.     

Evaluation of neutrophil acetyltransferase activity in patients with inflammatory disorders

The acetyltransferase activity of neutrophils from patients with inflammatory disorders was assayed using the homogenate preparation of neutrophils. The enzyme activity was evaluated on both non-stimulated and stimulated neutrophils. The enzyme activity of neutrophils from 21 patients with rheumatoid arthritis and 10 patients with Behcet's disease was not significantly different from that of the control group. In contrast, it was significantly elevated in patients with bacterial infection, especially that of non-stimulated cells. The increase in the enzyme activity best correlated with the degree of fever. The elevated enzyme activity tended to normalize during convalescence.     

Effect of radiotherapy on the neutrophil and the lymphocyte enzymatic equipment and serum immunoglobulins in patients with cancer of the larynx.

In 30 patients with cancer of the larynx, aged 40 to 70 years, treated by radiotherapy 6 to 9 years ago the decreased activity of neutrophil beta-glucuronidase and N-acetyl-beta-glucosaminidase accompanied by a decrease of absolute count of enzyme-positive cells was noted. Numbers of acid phosphatase-positive neutrophils were also decreased. Moderate increase of the neutrophil alkaline phosphatase activity and of numbers of enzyme-positive cells was another observed feature. The main finding in lymphocytes of the patients consisted in the appearance of cells exhibiting diffusion of lysosomal enzymes, especially of beta-glucuronidase, N-acetyl-beta-glucosaminidase and to a lesser degree of acid phosphatase into the cell cytoplasm. Moderate increase of immunoglobulin level, especially that of IgA, reflected probably the immunologic mobilization of patients.     

Acid phosphatase of neutrophils in patients with malignancies

In 132 patients suffering from various malignancies, including uterine carcinoma, breast cancer, gastric carcinoma, cancer of the colon, and additionally, patients with uterine myomas and endometriosis an increased activity of AP within the peripheral blood neutrophils could be stated by using a histochemical method. According to the author's opinion an increased activity of that enzyme reflects a response against local inflammatory processes frequently accompanying malignant tumours.     

The reactivity of the circulating neutrophils in human peripheral blood]

Using cytochemical, biochemical and disc-electrophoretic methods, the degree of extracellular secretion of peroxidase-containing neutrophil granules has been investigated as an index of their functional activity when in contact with antigens of extra- and intra-circulation. It was established that in in vitro contacts of neutrophils with alive and killed microbe culture St. aur., the quantity of the granules in the cells decreased as well as the enzyme activity in them. This is partially due to extracellular secretion of the granules content confirmed by the presence of peroxidase fractions in the solution. Similar results have been obtained for circulating neutrophils and serum of patients with acute pneumonia at the height of the disease. It is hold that antigen-induced extra- and intracellular neutrophil degranulation in the peripheral human blood reflects functional activity of the neutrophils.     

Urinary androgen- and estrogen excretion in men with pachydermia laryngis and cancer of the larynx.

Considering the larynx as a hormone dependent secondary sex characteristic has previously led to successful antiandrogentherapy of pachydermia of the vocal cords, which may constitute a precancerous state. As a first step to further evaluate the endocrine state of patients with precancerous lesions or cancer of the larynx, the urinary excretion of 17-hydroxysteroids, 17-ketosteroids, testosterone and estrogens has been determined in male patients with pachydermia laryngis (n = 15) or cancer of the larynx (n = 20) as compared to controls with different other otorhino-laryngological affections (n = 20). No difference between groups was found in 17-hydroxysteroids and no significant difference in 17-ketosteroid excretion. The pachydermia group as a whole showed significantly increased levels of testosterone (p = 0.01) and estrogen (p = 0.04) of 64.6 +/- 39.9 microgram/24 hr testosterone versus 34.7 +/- 19.3 microgram/24 hr in controls and 31.7 +/- 16 microgram/24 hr in laryngeal cancer and 277 +/- 14.8 microgram/24 hr total estrogens versus 19.1 +/- 12 microgram/24 hr and 17.8 +/- 8.1 microgram/24 hr respectively. These data further support the idea of hormonal factors playing an important role in the pathogenesis of pachydermia and thus possibly cancer of the larynx. So far, however, they do not permit definite conclusions on the pathogenetic mechanisms involved.     

Quantitative cytochemistry of blood neutrophils in myelodysplastic syndromes and chronic granulocytic leukaemia

Quantitative cytochemistry of components of blood neutrophil azurophilic granules (myeloperoxidase, chloroacetate esterase, beta-glucuronidase, and acid phosphatase) and specific granules (lactoferrin) has been performed by scanning and integrating microdensitometry in 13 patients with a myelodysplastic syndrome and 11 patients with chronic granulocytic leukaemia. Both patient groups showed a reduction of enzyme activity in azurophilic granules, and also of lactoferrin, consistent with abnormal development of neutrophil granules. These cytochemical changes in blood neutrophils are similar to those found in acute myeloid leukaemia, are consistent with a leukaemic maturation defect, and may be of diagnostic value.     

Inflammation and Cancer: Role of Annexin A1 and FPR2/ALX in Proliferation and Metastasis in Human Laryngeal Squamous Cell Carcinoma

The anti-inflammatory protein annexin A1 (ANXA1) has been associated with cancer progression and metastasis, suggesting its role in regulating tumor cell proliferation. We investigated the mechanism of ANXA1 interaction with formylated peptide receptor 2 (FPR2/ALX) in control, peritumoral and tumor larynx tissue samples from 20 patients, to quantitate the neutrophils and mast cells, and to evaluate the protein expression and co-localization of ANXA1/FPR2 in these inflammatory cells and laryngeal squamous cells by immunocytochemistry. In addition, we performed in vitro experiments to further investigate the functional role of ANXA1/FPR2 in the proliferation and metastasis of Hep-2 cells, a cell line from larynx epidermoid carcinoma, after treatment with ANXA1_2–26 (annexin A1 N-terminal-derived peptide), Boc2 (antagonist of FPR) and/or dexamethasone. Under these treatments, the level of Hep-2 cell proliferation, pro-inflammatory cytokines, ANXA1/FPR2 co-localization, and the prostaglandin signalling were analyzed using ELISA, immunocytochemistry and real-time PCR. An influx of neutrophils and degranulated mast cells was detected in tumor samples. In these inflammatory cells of peritumoral and tumor samples, ANXA1/FPR2 expression was markedly exacerbated, however, in laryngeal carcinoma cells, this expression was down-regulated. ANXA1_2–26 treatment reduced the proliferation of the Hep-2 cells, an effect that was blocked by Boc2, and up-regulated ANXA1/FPR2 expression. ANXA1_2–26 treatment also reduced the levels of pro-inflammatory cytokines and affected the expression of metalloproteinases and EP receptors, which are involved in the prostaglandin signalling. Overall, this study identified potential roles for the molecular mechanism of the ANXA1/FPR2 interaction in laryngeal cancer, including its relationship with the prostaglandin pathway, providing promising starting points for future research. ANXA1 may contribute to the regulation of tumor growth and metastasis through paracrine mechanisms that are mediated by FPR2/ALX. These data may lead to new biological targets for therapeutic intervention in human laryngeal cancer.     

Identification of elastase in human eosinophils: immunolocalization, isolation, and partial characterization.

Although an elastolytic activity in eosinophil-rich cell fractions from mice has been reported, this enzyme has not been purified and characterized as yet in any mammalian species. Eosinophilic elastase was isolated from human eosinophil fragments (cytosomes) obtained from normal and eosinophilic subjects. The enzyme was purified to apparent electrophoretic homogeneity by fast protein liquid chromatography. The enzyme shows the same physical properties of the major elastase isoenzyme of human neutrophils. In addition, like monocyte elastase, it reacts with a monoclonal antibody against human neutrophil elastase. The biochemical similarities observed between the above-mentioned enzymes and the immunolocalization findings strongly support the idea that human eosinophils and neutrophils contain the same enzyme activity. Eosinophils show immunoreactive material in both types of dense cytoplasmic granules. This observation supports the current hypothesis that the different types of eosinophilic granules represent successive morphological stages of maturation.     

Exocyclic malondialdehyde and aromatic DNA adducts in larynx tissues

Cigarette smoking and alcohol consumption, known to cause free radical generation and lipid peroxidation, are established risk factors for larynx cancer. Malondialdehyde (MDA) is a naturally occurring product of lipid peroxidation, capable of interacting with DNA to form exocyclic MDA-DNA adducts. In the present study, we investigated if the production of MDA-DNA adducts was increased in larynx cancer patients with respect to controls using (32)P-DNA postlabeling techniques. Moreover, we examined the potential effects of cigarette smoking and alcohol consumption on endogenous DNA adducts. We then analyzed the same set of larynx tissues for the presence of (32)P-postlabeled aromatic DNA adducts to determine more about the levels and types of adducts formed in the larynx. We observed that cancer patients tended to have increased levels of MDA and aromatic DNA adducts with respect to controls. In addition, smoking and alcohol were found to influence the formation of endogenous adducts in the larynx tissues. Finally, the amounts of endogenous adducts were found to be comparable to those observed for aromatic DNA adducts in the same set of larynx tissues. These findings imply that endogenous lesions, if not repaired, may contribute to larynx cancer development.     

The phenotype of peripheral blood neutrophils during the early stage of endometrial cancer

Peripheral blood neutrophils of 123 patients in stage Ia of the endometrial cancer have been assayed. The receptor system and ability of neutrophils to form extracellular traps (NET activity) were assessed by fluorescence microscopy, and spontaneous production of IL-2, IFN-γ, matrix metalloproteinase (MMP), G-CSF was found to exist determined with enzyme-linked immunosorbent assay. Phagocytic and myeloperoxidase activity, the level of cationic proteins, and neutrophil activity were evaluated in an NBT test determining the oxygen-dependent bactericidal action of neutrophils. Topology and rigidity of neutrophil membranes were assessed using scanning probe microscopy. It was established that an increased number of neutrophils was accompanied by changes of their receptor system, aerobic and anaerobic cytotoxicity, enhanced phagocytic activity, and reduced NET activity. The secretory activity of neutrophils changed. An increased level of MMP-1, possibly by enhanced production of reactive oxygen species, reduced IL-2 level, and a drastic increase in G-CSF were observed. The architectonics of neutrophils at stage Ia of endometrial cancer was characterized by altered cell shape and granularity loss. The rigidity of the cell membrane decreased. The changes in neutrophil morphology and persistent hyperactivity suggest the existence of the equilibrium between the immune system and the tumor in stage Ia of endometrial cancer.     

NADPH:O2 oxidoreductase of human eosinophils in the cell-free system

The NADPH oxidase of human eosinophils, measured in the cell-free system, shows the same characteristics as the enzyme from human neutrophils. All proteins required for activity of the enzyme are expressed in eosinophils at a higher level than in neutrophils. Eosinophils isolated from patients with chronic granulomatous disease show the same molecular defects as the neutrophils from these patients.     

Study of immune function of cancer patients influenced by supplemental Zinc or Selenium-Zinc combination

Since hair Zn and serum Zn are usually decreased in cancer patients, and Zn deficiency may reduce the function of T-cells, granulocytes, and Nk cells, we observed in cancer patients the influences of the Zinc or Selenium-Zinc on DNCB skin delayed hypersensitivity mediated by T cell, and the effects of Zinc on oxidative metabolic function of neutrophils and level of serum interferon that potentiate NK cell activity. The results showed that DNCB skin reaction was strengthened, the oxidative metabolic function of neutrophils and serum interferon level were increased by the drugs. It is reasonable to expect that Zinc or Selenium-Zinc is instrumental in restoring failing immunocompetence of cancer patient.     

Phospholipase C activity in human polymorphonuclear leukocytes: partial characterization and effect of indomethacin

Several hormones act at the cellular level to increase diacylglycerol via increased catabolism of phosphatidylinositol by phospholipase C. Diacylglycerol stimulates protein kinase C, leading to protein phosphorylation and hormone action. Since phospholipase C activity has not been well studied in man, we have established an assay for phospholipase C in human neutrophils. In this assay sonicates of neutrophils were incubated with L-3-phosphatidyl-[U 14C]-inositol and the incubation mixture extracted with chloroform/methanol. Following the additions of 2 mol/l KCl and chloroform, phospholipase C activity was determined by counting [14C] in the aqueous phase. The phospholipase C activity was linear with respect to time and the quantity of added enzyme. Optimum substrate concentration and pH were 2 mmol/l and 7.0, respectively. Optimal activity was dependent on Ca2+ (2 mmol/l) and deoxycholate (2 mmol/l). Naloxone, and PGD2, which affect various aspects of leucocyte function, had no significant effects on neutrophil PLC activity. The effects of various compounds with phospholipase A2 inhibitory activity were also tested on this enzyme. Of these, mepacrine, lidocaine and indomethacin inhibited the enzyme activity. The inhibition by indomethacin was of the noncompetitive type with an apparent Km of 0.17 X 10(-6) mol/l and apparent Ki of 3.6 X 10(-6) mol/l. From these data we conclude that indomethacin is capable of inhibiting phospholipase C activity in neutrophils at clinically significant levels and that this may be relevant in the therapeutic action of this drug.     

IL-8 stimulates phosphatidylinositol-4-phosphate kinase in human polymorphonuclear leukocytes.

IL-8 is a neutrophil-specific chemoattractant and cellular activator which exists in at least three forms, 69, 72, and 77 amino acids. The predominant monocyte product has 72 amino acids, whereas endothelial cells secrete the 77-amino acid form. The 72-amino acid form has been shown to increase intracellular calcium in neutrophils, but the exact biochemical pathways involved in stimulation of these cells is unknown. N-formyl peptide chemoattractants in neutrophils stimulate the formation of phosphatidylinositol-4,5-bisphosphate (PIP2), a reservoir for second messenger molecules and regulator of actin assembly through its association with the actin-binding proteins, profilin, and gelsolin. The present study examined whether IL-8 altered the enzyme which synthesizes PIP2, phosphatidylinositol-4-phosphate (PIP) kinase. Incubation of intact neutrophils with 10 nM IL-8 caused approximately a twofold increase in the activity of the enzyme. All forms of IL-8 stimulated PIP kinase activity in concentrations ranging from 1 to 50 nM, and the dose-response curves exactly correlated with the order of potency of these cytokines for interacting with the IL-8R on the surface of neutrophils. Lineweaver-Burk analysis of the kinetics of PIP kinase assayed in the presence of 0.03 to 0.7 mM ATP showed that 10 nM IL-8 increased the Vmax of the enzyme 38 to 70.5%, with no significant change in the apparent Km for ATP or for PIP. The stimulation of PIP kinase activity could not be explained by decreased degradation of PIP2 by phospholipase C or phosphomonoesterase activity in the membranes isolated from cells treated with IL-8 or by a decrease in the degradation of ATP. The microfilament disrupter, cytochalasin b, inhibited IL-8 induced stimulation of PIP kinase. These findings demonstrate that all forms of IL-8 stimulate PIP kinase in human neutrophils. This event may provide molecular signals to these cells that are necessary to maintain or change the state of microfilament assembly during cellular activation.     

Phagocytosis and peroxidase activity in neutrophils from peripheral blood of patients with malignant tumours of lung, stomach and large intestine

The phagocytic activity as revealed by latex ingestion and cytochemically demonstrated peroxidase activity of neutrophils from peripheral blood was investigated in 26 patients with pulmonary cancer, 22 patients with gastric cancer and 8 patients with tumours of large intestine and compared with results obtained in 40 healthy individuals. Statistically significant differences were found in case of both parameters between patients with pulmonary cancer and those with tumours of large intestine. The lowered phagocytic activity of neutrophils was accompanied by an enhanced peroxidase activity and vice versa. No statistically significant differences were observed, however, between the investigated groups of patients with cancer and the control group.     

The role of aromatic hydrocarbon hydroxylase in the pathogenesis of lung cancer

Aromatic hydrocarbons hydroxylase (AHH)--an enzyme of monooxydases group--catalyzes hydroxylation of polycyclic aromatic hydrocarbons yielding compounds of the direct cancerogenic properties. Inducibility determined genetically is a characteristic feature of AHH. Inducibility of AHH in patients with cancer of the lungs is higher than that in healthy population. According to some authors general population may be classified into three groups of : high, moderate, and low degree of AHH inducibility. A significant increase in the incidence of neoplasms related to an exposition to PAH was noted in patients with increased AHH inducibility. These neoplasms include: carcinoma of the lungs, mouth and larynx, cancer of the pharynx and leukoplakia. Moreover, the onset of these neoplasms is earlier than in subjects with moderate and low inducibility of AHH.