Activity of some lysosomal enzymes in peripheral blood lymphocytes of patients with lung cancer. A cytochemical study

In 33 patients with lung cancer (6 women and 27 men, aged at average 61.2 years) the activity and intracellular localization of acid phosphatase, beta-glucuronidase and N-acetyl-beta-glucosaminidase in peripheral blood lymphocytes were determined by means of semiquantitative cytochemical methods. In comparison to the control group of healthy subjects, the patients with lung cancer showed increased counts of acid phosphatase-positive lymphocytes with granular-diffuse cytochemical reaction, increased counts of beta-glucuronidase-positive lymphocytes with solely granular type of reaction and increased numbers of N-acetyl-beta-glucosaminidase-positive cells showing the granular, granular-diffuse and diffuse type of reaction. The total count of beta-glucuronidase-positive and N-acetyl-beta-glucosaminidase-positive lymphocytes was significantly elevated in these patients. The authors discuss the significance of their observations for evaluating lymphocyte response in patients with lung cancer.     

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.     

The state of immunity in the liquidators of consequences of the Chernobyl accident with cardio-vascular diseases

The purpose of the work was research and functional reserves immunity in participants in the Chernobyl with cardiovascular disease. A Clinical Lab 49 men aged 44 to 52 years with diseases of the cardiovascular system, participated in the Chernobyl nuclear power plant in 1986 to 1988. As control used data 33 patients with similar pathology, and 16 healthy men. Researched the total number of white blood cells, lymphocytes and transfusions, the absolute number and relative CD4+ and CD8+, CD 16, CD20+, CD95+ lymphocytes peripheral blood, number mononuclear, synthesizing IL-1beta, TNF-alpha, INF-alpha, IL-4, the content of lipids and proteins, the activity of alkaline phosphatase and mieloperoksidazy in neutrophils. To assess the functional reserve immune system blood samples studied people subjected to radiation doses 0.25, 50, 1.0 Gy of in vitro and studied the reaction cytochemical indicators neutrophils before and after the radiative forcing. The liquidators not detected significant changes in the absolute number of leucocytes, but compared with control groups noted significant reduction in the absolute number of CD8+ and CD20+ lymphocytes, increased the number of cells, expression of FAS-antigen, change the number of mononuclear spontaneously synthesizing and produce cytokines, decreased maintenance of cation proteins in neutrophils. Radiation samples peripheral blood liquidators caused the same reaction cytochemical indicators of neutrophils and control groups, the compensatory and adaptive nature of the changes in the immune system, developed in response to complex factors radiation accident.     

The lymphocyte cytochemical equipment and serum immunoglobulins in patients with precancerous states of the larynx.

In 24 men with precancerous states of the larynx, i.e. leukoplakia, papillomas and pachydermia, the peripheral blood lymphocytes were cytochemically stained for N-acetyl-beta-glucosaminidase, beta-glucuronidase, acid phosphatase and glycogen (PAS reaction). The results were expressed in terms of the absolute counts of enzyme- (or compound-) positive cells. The serum immunoglobulin IgG, IgA and IgM levels were also determined by Mancini's method. The results obtained were compared with those in 20 healthy men aged 20 to 30 years. It was found that the patients exhibited elevated numbers of N-acetyl-beta-glucosaminidase- and beta-glucuronidase-positive lymphocytes. A characteristic feature was an increase in the absolute counts of lymphocytes with diffuse and granular-diffuse types of cytochemical reaction for all enzymes studied. The number of cells with the granular type of enzymatic reaction (intact enzyme-positive lysosomes) was significantly diminished. These cytochemical alterations were accompanied by a significant increase in the serum IgA level. These results are discussed with reference to the lymphoid system response to tissues with precancerous lesions of the larynx.     

Digestion of the fifth component of complement by eosinophil lysosomal enzymes. Production of eosinophil specific chemotactic activity

Recently our laboratory has shown that neutrophils contain enzymatic activity within their lysosomal granules which will generate chemotactic activity for neutrophils and tumor cells from the fifth component of complement (C5). We have now expanded this initial observation and have demonstrated that eosinophils can release enzymatic activity from their lysosomal granules upon stimulation with immune complexes or opsoninized zymosan, but not with C5a or synthetic chemotactic peptides. Furthermore, the enzymatic activity released from the eosinophil lysosomal granules can cleave C5 into eosinophil-specific chemotactic activity. The generation of the eosinophil chemotactic activities from C5 is blocked by prior treatment of the eosinophil preparations with a number of protease inhibitors. The eosinophil-derived C5 cleaving activity possesses a pH optimum of 7.2, thus suggesting the enzymatic activity is a neutral protease. The demonstration that enzyme activities derived from eosinophils have the ability to generate eosinophil chemotactic factor(s) from C5 may explain why eosinophils are the predominant inflammatory cell in both nasal polyps and in the nasopharynx and bronchi of patients with allergic conditions such as hay fever and asthma.     

Immunological and cytochemical indices of white blood cells in old age

The group of aged subjects being 66 to 97 years old was compared with the middle-age group with regard to various immunological and cytochemical indices related to lymphocytes and neutrophils. The aged showed a lowered count and percentage of T cells, increased count and percentage of "non-B, non-T" lymphocytes, increased percentage of B cells. These alterations in the composition of lymphocyte subpopulation were associated with characteristic patterns of damage affecting the enzyme-positive lysosomal apparatus of lymphocytes with regard to acid phosphatase, beta-glucuronidase and N-acetyl-beta-D-glucosaminidase. There was a hundredfold smaller number of cells having intact enzyme-positive lysosomes in the aged than in the group of comparison. The changes mentioned above were also associated with the intracellular accumulation of glycogen in lymphocytes, decreased concentration of IgG and IgM in the serum and various changes in IgA concentration. Neutrophils of the aged were fewer in the blood of the aged than in younger subjects. However, an increased activity of myeloperoxidase, alkaline phosphatase, N-acetyl-beta-D-glucosaminidase, and an increased content of glycogen and lipids could be found in these cells. NBT-positive neutrophil numbers in the aged were lowered if the stimulated test was used and if there were no changes of the spontaneous test.     

Cytochemistry of nucleoproteids and some cathionic proteins in the peripheral blood leukocytes of patients with lung cancer.

In 40 patients with untreated lung cancer cytochemical studies of the peripheral blood leukocytes were conducted by means of a cytological method for the simultaneous staining of nucleoproteids (RNP and DNP) and some cathionic proteins (after Zvetkova and Zvetkov [60]). Changes were detected in the RNP cytoplasmic contents of lymphocytes, of which the most outstanding were the reduction and uneven distribution of RNP granules, their frequent extracellular expulsion by means of microclasmatoses, as well as changes in the staining of cathionic proteins of RNP accompanied by an increased nuclear chromatin condensation in the small and medium-sized lymphocytes. Parellel to reducing of the percentage of these cells in the peripheral blood of patients with advanced neoplastic disease an increased number of lymphoblastoid and monoblastoid cells is established with RNP diffusely stained, but reduced in quantity and localized in the cytoplasmic periphery and projections (compared to Downey type II atypical cells). By means of one of the variants of the method (modified type of Feulgen's reaction) a characteristic distribution and structuring of the nuclear chromatin is established in mono- and polymorphonuclear cells, most clearly expressed in the nuclei of monocytes and monoblastoid cells, as well as in nuclei of neutrophil granulocytes. In these cellular types a more specific nuclear modelling (microhypersegmentation) is observed resulting in multiple irregular nuclear projections on the nuclear surface, probably caused by subkaryolemal distribution of uneven chromatin thickenings. The changes are also recorded in the cathionic protein containing secondary cytoplasmic granules in granulocytes-neutrophils and eosinophils, probably associated with changes in the lysosomal and phagocytic functions of these cells in neoplastic diseases. The authors discuss the importance of the obtained results in connection with data on the participation of lymphocytes and neutrophils in the immune response to tumour antigenic stimuli during the course of the neoplastic process, as well as with data on the suppressive effect of antigenic (serum, viral) factors, possibly affecting the synthesis and the transport of cellular nucleoproteids (RNP and DNP) in leukocytes of cancer patients.     

The lysosomal enzymes of lymphocytes in the premature and the term infant.

Activity of acid phosphatase, beta-glucuronidase, and beta-glucosaminidase was determined in peripheral blood lymphocytes of 29 premature and 20 term infants with the use of cytochemical methods. The results were expressed semiquantitatively and included the total count of enzyme-positive and the enzyme-negative lymphocytes as well as the intracellular content of enzyme-positive and enzyme-negative lysosomal granules. The premature infant exhibited significantly lower activity of all the studied enzymes than the term infants. It thus argues in favour of the opinion that the lysosomal apparatus in lymphocytes undergoes development in the course of fetal maturation of the immune system. Evaluation of the activity of lysosomal enzymes in lymphocytes can serve as an indicator of fetal maturity and immunological status.     

Haematological data for Matsumoto Eosinophilic Shinshu rats as determined by an automated haematology analyser

The Matsumoto Eosinophilic Shinshu (MES) rat originated from an inbred mutant colony of rats with spontaneous eosinophilia. As part of an investigation of the pathogenesis of the MES rat, we examined the haematology data for 106 males and 88 females and age-associated changes using an automated haematology analyser, flow cytometric analysis and morphological examination. The data at 10 weeks of age showed the MES rats had higher counts for eosinophils and neutrophils, slightly higher counts for lymphocytes, monocytes, basophils, and large unstained cells (LUCs), and slightly lower values for the erythrocytic parameters when compared with Sprague-Dawley (SD) rats. In data for MES rats aged 8 to 20 weeks, eosinophil counts increased with age up to 20 weeks together with some increased neutrophil counts. After 11 weeks of age, counts for lymphocytes, monocytes, basophils, and LUCs in the MES rats were also slightly increased. In female MES rats, flow cytometric analysis showed increased counts for pan-T+ cells, but blasts, abnormal granulocytes and lymphocytes were not detected morphologically. The MES rat characterized by the haematological findings could be a useful animal model for studies of hypereosinophilia.     

Differential effect of growth factors on growth stimulation and phenotypic stability of glutamine-synthetase-positive and -negative hepatocytes in primary culture

In rat liver parenchyma, two subpopulations of hepatocytes can be distinguished by the absence or presence of the marker enzyme, glutamine synthetase (GS). Hepatocytes in the perivenous zone immediately adjacent to the hepatic venules in the liver acinus are positive for GS. Using autoradiography in combination with immunocytochemistry, the response of these two hepatocyte populations (GS positive and GS negative) to a variety of growth factors (defined compounds or complex stimuli) was investigated in vitro. Irrespective of the individual growth-promoting activity (which varied considerably), all stimuli led to much higher labeling indices in GS-negative cells as compared to GS-positive cells. In GS-negative cells, the strongest effect was exerted by serum obtained from partially hepatectomized rats (labeling index, 67%) and the conditioned media of JM1 and JM2 hepatoma cells (63%-82%), followed by a combination of insulin and either norepinephrine (46%) or epidermal growth factor (EGF; 42%). In contrast, serum had the weakest influence on GS-positive cells (0.3%), while the other potent stimuli enhanced the labeling index of these cells by between 6% and 15% within 48 h. The percentage of labeled nuclei was higher in mononucleated than in binucleated GS-positive hepatocytes. The time course of thymidine incorporation was also different for the two subpopulations. Under all growth-promoting conditions, the stimulation of GS-negative cells peaked between 72 and 96 h, while it increased continuously in GS-positive cells for at least 120 h, particularly in the case of serum. In proliferating cultures, both the absolute and the relative number of GS-positive hepatocytes decreased, while no such effect was found in various nonproliferating control cultures maintained at low and high cell density. Similar results were found for GS activity. In contrast, the hormonal induction of tyrosine aminotransferase (TAT) was not affected. It is suggested that these differences in the growth response of GS-positive and -negative cells contribute to the acinar gradient in hepatocyte proliferation that occurs during liver regeneration. Furthermore, the striking phenotypic instability of GS-positive cells that have undergone DNA synthesis and mitosis supports the hypothesis that cellular reprogramming depends on passage through the cell cycle.     

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.     

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.     

Activity of beta-glucuronidase in peripheral blood lymphocytes of patients with cancer of the larynx.

In 20 untreated male patients with cancer of the larynx, aged 35 to 55 years, the significant increase in the absolute count of beta-glucuronidase-positive lymphocytes in the peripheral blood was examined by means of the cytochemical method of Hayashi et al. (1964). The increase was due to an elevated absolute count of lymphocytes exhibiting the granular-diffuse and the diffuse enzymatic reaction; no significant changes were observed with regard to lymphocytes with the granular type of reaction. The authors discuss the significance of their observations for the evaluation of lymphocyte immune response against tumour specific antigens in patients with cancer of the larynx.     

Aromatase in the human testis

Low levels of testicular estrogen synthesis have been reported in a number of species, but the cellular localization has not been unequivocally established. To study aromatase in the human testis, we have combined immunocytochemistry with direct measurement of enzyme activity in the testicular 6μm cryosections. Thus, the functionality of the immunoreaction and its sensitivity can be assessed in quantitative terms. Testes were obtained from immediate autopsy from men aged 18–53 years, from surgery from two patients with prostatic cancer (67 and 74 years) and from two normal children aged 8 months and 3 years at autopsy. Benign testicular sex cord tumors were also examined from two unrelated patients aged 5 and 8 years with gynecomastia and diagnosed with Peutz-Jeghers syndrome. Our results consistently showed low to moderate staining intensity of immunoreactive aromatase in comparison to that of normal human placental cryosections. Immunoreactive aromatase was only present in the interstitial Leydig cells and absent from the Sertoli cells of all normal adult testes showing spermatogenesis. Aromatase activity correlated well with the intensity of the immunostain. However, there was no obvious relationship between the level of aromatase activity and increasing age. Generally higher levels were present in testes of young men (18–22 years). No immunostain in any cell type was detected in one 33-year-old patient with testicular cancer. In the testes of the two normal prepubertal boys, no immunostaining was observed. However, intensely stained Sertoli cells as well as high aromatase activity were observed in the testicular tumors of the patients with Peutz-Jeghers syndrome. Our results suggest that Leydig cells are the source of aromatase in normal men but that Sertoli cells may express this enzyme under abnormal conditions. The combined methods for measuring enzyme activity and immunoreactive aromatase are suitable for application to tissues expressing low levels of aromatase.     

The influence of tumor growth on natural cytotoxicity and activity of some lysosomal enzymes of human effector cells and the C3HA mouse splenocytes

In the course of malignant growth processes in patients with lung cancer, a decrease of natural cytotoxic activity of peripheral blood lymphocytes was observed. This process was accompanied by changes of activities of two lysosomal enzymes, arylsulfatase and acid phosphatase, suggesting participation of these enzymes in manifestation of effector functions of lymphocytes in cancer patients. The level of activity of granular enzyme, beta-glucuronidase, remained unchanged at all stages of disease. A study of natural killer activity of C3HA mice splenocytes after inoculation of transplantable hepatoma 22-a cells revealed a relative stability of the level of their cytotoxicity, and of the activities of lysosomal enzymes--arylsulfatase, acid phosphatase, alpha-mannosidase, acid lipase, N-acetyl-beta-D-glucosidase, and beta-galactosidase, beginning from the 3rd day after hepatoma implantation.     

Ultrastructure of human dermal mast cells in 29 different lysosomal storage diseases

The effect of lysosomal storage diseases on the ultrastructure of human mast cells has not previously been reported. Indeed, there has been little published evidence indicating that mast cells contain typical lysosomes. However, mast cell cytoplasmic granules contain hydrolases similar to those found in lysosomes, but which differ from lysosomal hydrolases in exhibiting optimal activity at higher pH. We therefore examined by transmission electron microscopy the dermal mast cells in 58 biopsies of patients exhibiting 1 of 29 different lysosomal storage diseases. We found mast cells containing abnormal lysosomes in 16 of these disorders. In 6 of these 16 diseases, the mast cells' cytoplasmic granules appeared normal. These observations indicate that human mast cells can contain lysosomes, and provide evidence that the enzymes affected by lysosomal storage diseases are active in mast cells.     

Influence of β-endorphin on the proliferative and phagocytic activities of peripheral blood cells of men and women from different age groups

It has been found that β-endorphin modulation of lymphocyte proliferative activity in male donors is mainly observed at a relatively young age (in groups aged 20–29 and 30–39 years), it gradually becomes lower with age, and disappears in donors at aged 50–60 years. At the same time, women have a prolonged modulating effect of peptide on proliferation. In women aged 50–59 years, the peptide has a marked promotional effect on spontaneous proliferation at concentrations of 10^?7, 10^?8, and 10^?10 M induced by a suboptimal concentration of phytohemagglutinin (PHA) at 10^?10 M, while in women aged 30–39 years, β-endorphin suppresses PHA-induced proliferative response. In men aged 20–29 years, β-endorphin stimulates the uptake capacity of neutrophils, whereas in those aged 50–59 years, this capacity is suppressed by β-endorphin. In female donors from any age groups, β-endorphin was not found to influence the activity of neurophils.     

Cytologic and immunocytochemical studies of cerebrospinal fluid in meningeal sarcoidosis. A case report.

Serial studies were done on cerebrospinal fluid (CSF) from a patient with sarcoidosis involving the meninges. Initially when the disease was active, the CSF protein was increased and glucose decreased. The number of cells in the CSF was moderately increased, and many mononuclear cells were present. Cytologic studies of the CSF showed many normal and some atypical lymphocytes. Immunochemical studies showed that most of these lymphocytes were T cells, with T-helper cells predominating over T-suppressor cells by a ratio of 3.92; B-lymphocytes were polyclonal. Subsequent studies of the CSF over the following three and one-half years showed that the protein and glucose content and the cell counts in the fluid did not correlate well with the activity of the disease. The number of atypical lymphocytes seemed to be a more useful marker of disease activity in the patient. Cytologic studies, when interpreted within the context of other CSF and clinical findings, are useful for the assessment of patients with sarcoidosis involving the meninges.     

Morphometry of feline neutrophil granule genesis

During neutrophil granule genesis, the formation of primary granules is generally thought to be limited to the promyelocyte stage; whereas synthesis of secondary granules is thought to occur only at the myelocyte stage. This hypothesis was tested morphometrically in feline neutrophils that are known to contain both granule types. Marrow specimens obtained from six cats were stained with peroxidase for identification of neutrophil primary granules and counterstained with periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) for identification of secondary granules. By regression analysis using arithmetic models, numbers of cytoplasmic granules in 311 cells were correlated with the degree of nuclear chromatin condensation, which was shown to be an adequate parameter for cell maturation. Promyelocytes and myelocytes had similar mean numbers of peroxidase-positive granules per unit area. A significant increase (p less than or equal to 0.0001) in the numbers of peroxidase-positive granules was noted between the metamyelocyte and the mature neutrophil stage, despite the lack of peroxidase activity in endoplasmic reticulum and Golgi lamellae. By contrast, a significant increase of peroxidase-negative granules between the metamyelocyte and the mature neutrophil stage was not clearly established with these methods. The increase in peroxidase-positive granules may indicate continued production of peroxidase-containing granules and/or redistribution of peroxidase among lysosomal organelles in late feline neutrophils.