pH effects on cystine transport in lysosome-rich leucocyte granular fractions.

Inhibitors of lysosomal acidification (4,4'-di-isothiocyanostilbene-2,2'-disulphonate, NN'-dicyclohexylcarbodi-imide, carbonyl cyanide m-chlorophenylhydrazone, NH4Cl and methylamine hydrochloride) did not alter cystine egress or countertransport in polymorphonuclear-leucocyte lysosome-rich granular fractions at pH 7.0. Together, 2 mM-MgCl2/MgATP and 90 mM-KCl stimulated cystine egress 2-fold, but this effect also was not influenced by inhibitors of ATP-dependent lysosomal acidification. MgCl2/MgATP stimulated cystine transport at pH 5.5, but the effect also occurred with MgCl2, MgSO4 or MnCl2 alone, was prevented by chelation, and was not seen with NaATP; therefore, it was considered a bivalent-cation, not an ATP, effect. Proton-pump-mediated acidification of lysosomes does not appear to be required for cystine transport in normal polymorphonuclear-leucocyte granular fractions, as reported for lymphoblast lysosomes.     

Subpopulation analysis of human granular lymphocytes: associations with age, gender and cytotoxic activity

Levels of human blood granular lymphocyte subpopulations were enumerated in 105 healthy donors ranging in age from 0 to 79 years. Using double-label immunofluorescence, subpopulations of granular lymphocytes were enumerated as follows: Leu7+Leu2+, Leu7+Leu3+, Leu7+11-, Leu7+77+ and Leu7-Leu11+. The proportion of cells with granular lymphocyte morphology was determined by Giemsa staining. Natural killer (NK) cell activity against K-562 cells and lymphokine-activated killer activity against non-cultured melanoma cells were examined in parallel. Levels of total Leu7+ and Leu11+ cells increased with age (p = 0.0001) and were higher in males than females (p = 0.001). The total number of cells with granular lymphocyte morphology had an age-related increase (p = 0.001), but were not significantly higher in males than in females (p = 0.07). There was no selective increase in one granular lymphocyte subpopulation versus another since the Leu7+Leu11- (p = 0.0001), the Leu7+Leu11+ (p = 0.0001), the Leu7+Leu2+ (p = 0.0001) and the Leu7+Leu3+ (p = 0.0004) all had similar age-related increases. The one exception was the Leu7-Leu11+ (p = 0.1) granular lymphocyte subset which was low in the first decade of life but had reached maximum levels in the second decade. NK cell activity against K-562 cells was moderately increased with age (p = 0.06) with males and females exhibiting comparable activity. In contrast. lymphokine-activated killer cytotoxicity of non-cultured melanoma cells was similar in all age groups. NK cell activity was highly correlated with levels of morphologically defined granular lymphocytes (p = 0.005) and moderately with total Leu11+ cells (p = 0.06) but not with other subpopulations of granular lymphocytes.     

Axillary granular parakeratosis

Axillary granular parakeratosis is an alteration of keratin characterized by a thick parakeratotic horny layer with abundant intracellular keratohyalin granules. It was first described in 1991 and since then 32 cases have been reported from USA, Europe and Australia. Lesions may affect intertriginous areas other than the axilla. The disease has apparently not been previously described in Latin America. Three overweight Colombian women were diagnosed with axillary granular parakeratosis. They presented encrusted, hyperkeratotic, hyperpigmented and pruriginous papules and plaques which affected both axillae in two women and only one in the other. Lesions had persisted for two and four months in two patients and for one year in the third. Clinical diagnoses were benign familiar pemphigus and tinea nigra. Skin biopsies showed a thick parakeratotic basophilic horny layer. Electron microscopy demonstrated a high content of keratohyalin granules. No Langerhans cells were demonstrated in the lesions using IHC for S-100 protein. No fungi were seen with the PAS stain. Infundibula showed thick horny plugs with changes similar to those seen in the epidermis. Dermal tissue showed few perivascular lymphocytes. These findings suggest that the disease has an irritative pathogenesis. Clinical histories indicated that the three women were overweight and used several types of antiperspirants. These factors plus local irritation and humidity apparently triggered the keratinization response.     

Crohn’s disease of the colon: ultrastructural changes in submuscular interstitial cells of Cajal

Interstitial cells of Cajal (ICC) at the submuscular border of the human colon (ICC-SMP) are the proposed pacemaker cells of the musculature. In patients with Crohn’s disease (CD) of the colon, ICC-SMP showed characteristic cytological changes from controls. The changes comprised secondary lysosomes in connection with lipid droplets and cytoplasmic vacuoles or multiple empty, confluent and often outbulging vacuoles merging with cisterns of granular endoplasmic reticulum and clusters of glycogen granules. These changes were most pronounced in patients with macroscopical mucosal inflammation but were also demonstrable in uninvolved colonic segments. Relationships of ICC to other cells were undisturbed. The changes were selective to ICC-SMP, as glial cells, muscle cells and fibroblast-like cells at the submuscular border showed no cytological alterations compared with controls. Varicosities of the submuscular plexus were often empty and dilated. Fibroblast-like cells selectively encased macrophages and mast cells. The cytological changes in ICC-SMP in CD are thus similar to changes seen in ulcerative colitis and may be of pathophysiological significance with regard to the motility and sensory disturbances seen in patients with CD.     

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.     

The ultrastructure of pinealocytes in the golden mole (Amblysomus hottentotus) with special reference to the granular vesicles

Summary The ultrastructure of the pinealocytes of the golden mole (Amblysomus hottentotus), a blind subterranean mammal living in complete darkness, was examined and compared with that of pinealocytes of other mammals. On the basis of the presence of granular vesicles and glycogen granules, only one population of pinealocytes was found. Large mitochondria, ribosomes, lipid inclusions and scarce lysosomes were observed in the perikaryon. Numerous glycogen granules, often forming typical accumulations, were frequently found to be associated with typical vacuoles. An extraordinary large number of granular vesicles is characteristic of the golden mole pinealocyte. Phenomena of excretion of compound(s) stored in the granular vesicles were identified. Morphologically this is characterized by dissociation of its granular core in situ while its limiting membrane is preserved. The physiological significance of the secretory process characterized by the formation of these granular vesicles is discussed.A portion of these observations were presented at the International Symposium on the Pineal Gland, Jerusalem, Israel, November 14–17, 1977     

Comparison of T cell receptor gene rearrangements in patients with large granular T cell leukemia and Felty's syndrome

Felty's syndrome (FS) refers to the occurrence of rheumatoid arthritis, splenomegaly, and neutropenia. A subset of these patients has recently been described with a chronic T cell leukemia of large granular lymphocytes (LGCL). To examine the spectrum of lymphocyte abnormalities in FS and LGCL, we examined phenotypic and genotypic properties of lymphocytes from eight FS patients. In two of these FS patients, we observed an elevated proportion of T cells with an unusual phenotype (CD3+/Leu-7+/Leu-8-/CR3+) (46 +/- 5% of mononuclear cells). The FS lymphocytes had large granular morphology on Wright-Giemsa stain and were active in antibody-dependent cellular cytotoxic activity. This phenotype, morphology, and activity was similar to LGCL patients except that the latter T cells additionally expressed the Fc-IgG receptor recognized by monoclonal antibody Leu-11 (CD 15). In the remaining six FS patients, the proportion of CD3+/Leu-7+/CR 3+ T cells was only 10 +/- 8%, which was not significantly different from age-matched normal subjects (6.6 +/- 2.2%). To determine the clonality of T lymphocytes in FS and LGCL, we examined DNA for rearrangements of the T cell antigen receptor beta-chain (Ti beta) and gamma-chain (Ti gamma) genes by using Southern blotting techniques. We found a clonal rearrangement of the Ti beta 1 and Ti gamma genes in both LGCL patients. In contrast, no clonal rearrangements of Ti beta or Ti gamma genes were detected in lymphocytes from the FS patients. These results indicate that FS patients are heterogeneous in their phenotype and that one subset exhibits polyclonal expansion of an unusual lymphocyte subset.     

Increased number of functionally defective large granular lymphocytes in lymphoma patients

50 untreated patients with either Hodgkin's disease or non-Hodgkin lymphoma were studied for natural killer (NK) activity on the single cell level. The non-Hodgkin lymphoma patients had a significantly higher proportion of large granular lymphocytes-cells mediating the natural cell mediated cytotoxicity in humans-among peripheral blood lymphocytes. Yet, the single cell assay in agarose and the standard 51Cr assay revealed a significantly decreased capacity to bind and lyse the K562 target cells. The recycling capacity was also found to be lower in the lymphoma patients' NK cells compared to the healthy controls.     

Frequency of large granular lymphocytes in peripheral blood of healthy persons and breast cancer patients

Summary The frequency of large granular lymphocytes (LGL) in the peripheral blood of healthy persons (n=56) and breast cancer patients (31 cases with stage-I and -II disease and 42 cases with stage-III and -IV disease) was studied. The frequency of LGL in peripheral blood was significantly depressed in cancer patients, and particularly in patients with advanced breast cancer.     

Frequency of large granular lymphocytes in peripheral blood of healthy persons and breast cancer patients

The frequency of large granular lymphocytes (LGL) in the peripheral blood of healthy persons (n = 56) and breast cancer patients (31 cases with stage-I and -II disease and 42 cases with stage-III and -IV disease) was studied. The frequency of LGL in peripheral blood was significantly depressed in cancer patients, and particularly in patients with advanced breast cancer.     

Occurrence of immunoreactivity for adipocyte-type fatty acid binding protein in degenerating granulosa cells in atretic antral follicles of mouse ovary

The localization of adipocyte-type fatty acid binding protein (A-FABP) in the mature mouse ovary was examined by immuno-light and electron microscopy. Solitary round cells showing the distinct immunoreactivity for A-FABP were detected in 1–6 antral follicles. In sets of two consecutive sections in a mirror alignment on slide glasses which were treated for immunoreactivity for A-FABP and TUNEL reaction separately, cells immunoreactive for A-FABP appeared in the same antral follicles as containing cells exhibiting TUNEL-reaction. In immunoelectron microscopy, A-FABP-immunopositive cells were found to contain highly electron-dense nuclei of round, irregular or crescent shapes together with cytoplasmic remnants without any features of macrophages or cells of extrinsic origin. Therefore the cells were identified as apoptotic granulosa cells. The apoptotic cells immunoreactive for A-FABP were often seen to be enclosed/engulfed in adjacent cells exhibiting normal ultrastructures without containing numerous lysosomes. The present findings suggest that A-FABP is involved in the apoptosis of ovarian granulosa cells, probably through its interaction with peroxisome proliferator activated receptors.     

Preoperative diagnosis of a mediastinal granular cell tumor by EUS-FNA: a case report and review of the literature

We report the first case of a posterior mediastinal granular cell tumor initially diagnosed on cytologic material obtained via endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) in a 51-year-old male with a prior history of colon cancer. Aspirates obtained were cellular and composed of polygonal cells with abundant granular cytoplasm and small, round dark nuclei. An immunoperoxidase stain performed on the cell block for antibodies to S-100 protein showed strong, diffuse staining of the cytoplasmic granules. Electron microscopy performed on the cell block revealed numerous cytoplasmic lysosomes. This is the first case report in the English literature of a definitive preoperative diagnosis of a mediastinal granular cell tumor utilizing material obtained via EUS-FNA.     

Chemotactic effect of human recombinant interleukin 2 on mouse activated large granular lymphocytes

Human recombinant interleukin 2 (hrIL-2) was demonstrated in vitro to be chemotactic for mouse large granular lymphocytes (LGL) activated in vivo by virus infection. Peritoneal exudate cells harvested from virus-infected mice were used as a source of LGL. LGL collected from mouse hepatitis virus-infected mice at 3 days postinfection were a source for NK 1.1 positive natural killer (NK)/LGL. LGL collected from mice treated with antiserum to gangliotetraosylceramide and infected with lymphocytic choriomeningitis virus for 7 days were used as a source for Lyt-2 positive cytotoxic T lymphocytes (CTL)/LGL. Both NK/LGL and CTL/LGL responded chemotactically to hrIL-2, purified IFN-beta, and to crude cell-free washout fluids collected from the peritoneal cavity of virus-infected mice. hrIL-2 had chemotactic activity for virus-elicited granular and agranular lymphocytes but did not attract the contaminating macrophages, in contrast to IFN-beta, which displayed chemotactic activity for virus-elicited granular and agranular lymphocytes as well as macrophages. The migration to hrIL-2 was inhibited by a monoclonal antibody (7D4) to the IL-2 receptor, but treatment with 7D4 did not affect migration in response to IFN-beta. Microscopic examination of Wright's-Giemsa-stained migrated NK/LGL and CTL/LGL revealed that the majority of migrated LGL in either LGL population had a blast cell morphology (enlarged cells with rich basophilic cytoplasm). The frequency of cells bearing the LGL morphology within the virus-elicited nonadherent peritoneal exudate cell population was on incubation in vitro, stabilized by either hrIL-2 or IFN-beta. These data suggest that another important immunomodulating function of IL-2 may be to attract activated NK/LGL and CTL/LGL to sites of inflammation.     

Gratuitous dechlorination of chloroethanes by methanogenic granular sludge

The dechlorinating activity of a methanogenic granular sludge from a methanol-fed upflow anaerobic sludge blanket reactor was investigated with chlorinated ethanes. This unadapted methanogenic consortium degraded all chloroethanes tested. The product formation rates decreased with the number of chlorine substituents. The more highly chlorinated ethanes were also converted, although at a lower rate, in the presence of autoclaved (dead) sludge, indicating the involvement of reduced heat-stable cofactors like vitamin B₁₂ and F₄₃₀. Direct chemical dechlorination of hexa-, penta- and tetrachloroethanes was also observed in medium without sludge, although at a much lower rate. The results show the importance of cometabolic and abiotic (chemical) conversions for the transformation of chlorinated ethanes by the methanogenic consortium. The types of reaction and the products formed were correlated with the Gibbs free-energy change (ΔG ^0′). Reductive hydrogenolysis and dichloroelimination were important dechlorinating mechanisms. Generally, these reactions have a higher ΔG ^0′ value than dehydrochlorination reactions, which occurred less frequently during the transformation of chloroethanes by the methanogenic granular sludge. Received: 8 June 1998 / Received revision: 7 September 1998 / Accepted: 13 September 1998     

Characteristics of cystine counter-transport in normal and cystinotic lysosome-rich leucocyte granular fractions.

Normal leucocyte lysosome-rich granular fractions exhibited counter-transport of cystine, confirming that cystine transport across the lysosomal membrane is carrier-mediated. The trans-activation of cystine transport was temperature-dependent but relatively independent of the external Na+ or K+ concentration in phosphate buffer. Counter-transport, measured as uptake of exogenous [3H]cystine, increased with increasing intralysosomal cystine content up to approx. 3 nmol of half-cystine/unit of hexosaminidase activity. The amount of [3H]cystine entering lysosomes loaded with unlabelled cystine decreased when unlabelled cystine was added to the extralysosomal medium. Lysosomal cystine counter-transport was stereospecific for the L-isomer. Cystathionine, cystamine and cysteamine-cysteine mixed disulphide gave evidence of sharing the lysosomal cystine-transport system, although at lower activity than cystine. Other tested amino acids, including arginine, glutamate and homocystine, were inactive in this system. Nine leucocyte lysosome-rich preparations from eight different cystinotic patients displayed virtually no counter-transport of cystine, conclusively establishing that a carrier-mediated system for cystine transport is dysfunctional in cystinotic lysosomes.     

The tumor-bearing state induces augmented responses of organ-associated lymphocytes to high-dose interleukin-2 therapy in mice

 A high-dose bolus regimen for interleukin(IL)-2 administration to cancer patients frequently causes serious side-effects in which various organs are involved. In order to reveal the mechanism of toxicities associated with this regimen, we compared the augmenting effect of high-dose IL-2 on murine organ-associated lymphocytes between neoplastic and non-neoplastic states. Intraperitoneal administration of IL-2 at a dose of 10⁵ JRU (Japanese Reference Units) twice daily for 3 days led to the death of all the syngeneic MH134-hepatoma- or X5563-myeloma-bearing mice, whereas it had no lethal effect on non-tumor-bearing mice. Histological and morphometric analyses demonstrated that tumor-bearing mice displayed more extensive infiltration of large granular lymphocytes and agranular lymphocytes in the liver and lungs than did the non-tumor-bearing mice. Large granular lymphocytes had the ultrastructural characteristics of lymphokine-activated killer cells. Lymphocytes often underwent extravasation into the interstitial space and exhibited local proliferation without causing any direct injury to apposed parenchymal cells. Flow-cytometric analysis of hepatic mononuclear cells demonstrated that IL-2-receptor-β(IL-2Rβ)-bearing lymphocytes, i.e., natural killer cells and intermediate CD3 cells, were increased in number in the neoplastic state before the IL-2 injection. The present study indicates that the tumor-bearing state increases the number of organ-associated IL-2Rβ⁺ lymphocytes, which are then greatly amplified by the challenge of high-dose IL-2, leading to the functional disturbance of organs. We have further demonstrated here that an intermittent low-dose IL-2 regimen has a potential therapeutic effect on tumor regression without causing lethal side-effects. Received: 15 July 1996 / Accepted: 23 July 1997     

Prolonged survival of AVN wistar rats with transplanted yoshida sarcoma and increase of granular lymphocytes after administration ofBacillus firmus and their crude lipids

Bacillus firmus is a Gram-positive, aerobic, sporulating, nonpathogenic air contaminant which, according to earlier findings, is a strong polyclonal activator of B lymphocytes. The crude lipids of this microbe induced significant resistance of mice against listerial infection. The administration of bacterin, like that of crude lipids obtained by the extraction of cell suspension with chloroform—methanol to rats, strain AVN Wistar, transplanted later with Yoshida sarcoma, significantly prolonged the survival of the animals in comparison with the control group. At the same time the number of granular lymphocytes was increased. The destruction of tumor cells in the peritoneal exudate of immunostimulated rats was also determined. Dedicated to Professor J. Šterzl on the occasion of his 70th birthday     

Chemotaxis of large granular lymphocytes

The hypothesis that large granular lymphocytes (LGL) are capable of directed locomotion (chemotaxis) was tested. A population of LGL isolated from discontinuous Percoll gradients migrated along concentration gradients of N-formyl-methionyl-leucyl-phenylalanine (f-MLP), casein, and C5a, well known chemoattractants for polymorphonuclear leukocytes and monocytes, as well as interferon-beta and colony-stimulating factor. Interleukin 2, tuftsin, platelet-derived growth factor, and fibronectin were inactive. Migratory responses were greater in Percoll fractions with the highest lytic activity and HNK-1+ cells. The chemotactic response to f-MLP, casein, and C5a was always greater when the chemoattractant was present in greater concentration in the lower compartment of the Boyden chamber. Optimum chemotaxis was observed after a 1 hr incubation that made use of 12 micron nitrocellulose filters. LGL exhibited a high degree of nondirected locomotion when allowed to migrate for longer periods (greater than 2 hr), and when cultured in vitro for 24 to 72 hr in the presence or absence of IL 2 containing phytohemagluttinin-conditioned medium. The chemotactic LGL was HNK-1+, OKT11+ or HNK-1+, OKT11- on the basis of monoclonal antibody and complement depletion. They did not bear either T cell or monocyte cell surface markers, exhibiting an OKT3-, OKT4-, OKT8-, OKM1-, and MO2- phenotype, and did not form E rosettes at 29 degrees C, which is characteristic of lytic NK cells in contrast to T cells. Furthermore, a rat LGL leukemia (RNK) exhibited a chemotactic response to both f-MLP and casein. LGL chemotaxis to f-MLP could be inhibited in a dose-dependent manner by the inactive structural analog CBZ-phe-met, and the RNK tumor line specifically bound f-ML[3H]P, suggesting that LGL bear receptors for the chemotactic peptide.     

Biodegradation of azo dyes in cocultures of anaerobic granular sludge with aerobic aromatic amine degrading enrichment cultures

A prerequisite for the mineralization (complete biodegradation) of many azo dyes is a combination of reductive and oxidative steps. In this study, the biodegradation of two azo dyes, 4-phenylazophenol (4-PAP) and Mordant Yellow 10 (4-sulfophenylazo-salicylic acid; MY10), was evaluated in batch experiments where anaerobic and aerobic conditions were integrated by exposing anaerobic granular sludge to oxygen. Under these conditions, the azo dyes were reduced, resulting in a temporal accumulation of aromatic amines. 4-Aminophenol (4-AP) and aniline were detected from the reduction of 4-PAP. 5-Aminosalicylic acid (5-ASA) and sulfanilic acid (SA) were detected from the reduction of MY10. Subsequently, aniline was degraded further in the presence of oxygen by the facultative aerobic bacteria present in the anaerobic granular sludge. 5-ASA and SA were also degraded, if inocula from aerobic enrichment cultures were added to the batch experiments. Due to rapid autoxidation of 4-AP, no enrichment culture could be established for this compound. The results of this study indicate that aerobic enrichment cultures developed on aromatic amines combined with oxygen-tolerant anaerobic granular sludge can potentially be used to completely biodegrade azo dyes under integrated anaerobic/aerobic conditions. Received: 16 September 1998 / Received revision: 14 December 1998 / Accepted: 21 December 1998     

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.