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.     

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.     

The number and ultrastructure of CD4+ lymphocytes in the blood of healthy persons and in patients with type 1 diabetes mellitus

The flowing cytofluorometric and electron microscopic methods have been used to study the content and ultrastructure of blood CD4 lymphocytes in healthy and type I diabetic patients. Analysis of submicroscopic structure of the lymphocytes expressing CD4 antigen has revealed its morphological heterogeneity. It has permitted distinguishing four groups of cells. A decrease of the content of CD4+ lymphocytes at the expense of the first group cells and relative increase in the number of the fourth-group cells are observed in the majority of diabetic patients. The availability of parallel-tubular structures in granules forming clusters and first-group lymphocytes, an increase in the number and size of granules of the fourth-group are the most typical changes of ultrastructural characteristics.     

青少年食蟹猴糖尿病模型的肝脏病理生理学研究

目的通过对胰岛素用量不足条件下链脲佐菌素诱导的青少年食蟹猴1型糖尿病模型肝脏病理生理学的研究,探讨长期高血糖所致青少年食蟹猴肝损伤特点及机制。方法通过静脉注射68 mg/kg的链脲佐菌素,诱导4只3岁的食蟹猴成为1型糖尿病模型,然后经长期的血糖监测和静脉糖耐量实验来评价该模型的可靠性及稳定性,造模4年后,对模型猴进行血生化、PAS染色、苏丹III染色及普通病理和超微病理等指标的检测,另外选取4只健康与模型猴年龄匹配的猴作为正常对照组,同时进行相应的检测。结果与正常对照组比较,糖尿病猴血清学检测指标中总胆汁酸、尿素氮、谷丙转氨酶、谷草转氨酶、胆碱酯酶、乳酸脱氢酶、总胆固醇、甘油三酯和低密度脂蛋白胆固醇明显升高。组织化学染色结果显示,与正常猴比较,糖尿病猴中央静脉区肝实质细胞肿胀,肝细胞PAS染色（糖原染色）加深,苏丹Ⅲ染色（脂肪染色）阳性细胞增多;电镜结果显示糖尿病猴肝细胞内胞质糖原颗粒增多;线粒体电子密度显著增高,结构不清;窦周隙内含有大量脂滴的肝星状细胞明显增多。结论在长期胰岛素用量不足血糖控制不理想的条件下,青少年食蟹猴1型糖尿病模型肝脏特异性的病理改变是肝糖原贮积和含有大量脂滴的肝星状细胞增生,这些病理改变与非酒精性脂肪肝病的病变特点存在显著不同,但其机制目前尚不清楚。     

长吻鮠血细胞发生的研究

用Wright-Giemsa和PAS染色对长吻鮠头肾、肾脏、脾脏、肝脏等器官组织的涂片、印片染色观察发现,头肾、肾脏和脾脏是其主要造血器官。红细胞、粒细胞和淋巴细胞主要在肾脏和头肾中发生,其次是脾脏。单核细胞则主要在肾脏和脾脏中发生,头肾中也有少量单核细胞产生。肝脏中无原始型血细胞,可能不是其造血器官。红细胞的发育经历四个阶段,其胞体体积经历了由大到小,由小到大再变小的"两大两小"发育过程;粒细胞的发育经历五个阶段,其胞体体积均由大变小,双叶或多叶核的粒细胞可能是衰老的粒细胞亦即核的分叶是粒细胞衰老的标志;淋巴细胞和单核细胞的发育各经历了三个阶段,两者发育成熟过程中胞体体积均由大变小。巨噬细胞由单核细胞发育而来。原血细胞和部分早期幼稚血细胞可以进行有丝分裂,部分成熟红细胞和血栓细胞可以进行直接分裂。红细胞在整个发育过程中,PAS反应均呈阴性,各类白细胞的发育过程中,PAS反应由阴性到阳性并逐渐增强,这显示随着白细胞的逐渐发育成熟,细胞内糖原物质含量逐渐增多。     

Mathematical simulation of the regulation of the size of the lymphoid population in relation to chronic lymphoid leukemia

A model was constructed of accumulation kinetics of labeled lymphocytes based on the experiments of long-term injection of 3H-thymidine label in vivo into the blood of healthy and suffering from chronic lympholeucosis animals. There was found an essential difference between the coefficients of reproduction and death of cells of the proliferating pool for the normal, initial and advanced stages of the disease. This served as a basis for the creation of the closed non-linear model of autoregulation of lymphoid population size describing different stages of leucosis development.     

过敏性紫癜兔模型的免疫学改变及机制初探

目的通过对过敏性紫癜兔模型免疫学改变的初步研究,探讨该病的免疫学发病机制。方法通过对过敏性紫癜兔模型进行血常规检测、ELISA方法检测免疫细胞及细胞因子CD3、CD4、CD8、CD4/CD8、IL-2、TNF-α含量;免疫荧光方法检测皮肤、肾脏免疫球蛋白IgA,IgG及补体C3;肾小球Masson染色、PAS染色;皮肤、肺组织的Luna染色等,并与对照组进行比较分析。结果与对照组相比,模型组兔血白细胞（WBC）增多,中性粒细胞（NEU）及百分比增高,嗜酸性粒细胞（EOS）及百分比增高,嗜碱性粒细胞增多等,差别均具有统计学意义;外周血CD3＋T淋巴细胞含量减少,CD4＋T淋巴细胞含量减少,CD8＋T淋巴细胞含量增多,CD4＋/CD8＋比值下降,细胞因子IL-2水平下降,TNF-α水平升高,差别均具有统计学意义;皮肤、肾脏免疫球蛋白IgA、IgG、C3表达增多;肾小球胶原纤维增生,系膜增厚,系膜基质增多;皮肤真皮层及肺组织内嗜酸性粒细胞表达增多。结论将为明确其发病机制,临床诊断和疗效观察找出新的指标,选择适当的治疗方案提供有价值的理论依据。     

A histochemical study on the snout tentacles and snout skin of bristlenose catfish Ancistrus triradiatus

Histological sections of the tentacles of Ancistrus triradiatus revealed that they contain many goblet cells with granule-like sections distributed along the edge of the tentacles (mean 302 cells mm⁻¹). Various histochemical methods were used to characterize the contents of the goblet cells. The results indicated that the contents were periodic acid-Schiff (PAS) positive, glycogen negative and no sulphated mucins could be found. The contents of the goblet cells were acid mucins and O-acetylated sialomucins which had high energy content. The snout skin of the male also contained goblet cells with higher density (mean 755 cells mm⁻¹) than that of the tentacles. Snout skin of female A. triradiatus , on the contrary, did not contain any goblet cells. Instead, many minute spines could be found. Based on the acid mucins and O-acetylated sialomucins secretions of goblet cells which are of high nutritional values, it is hypothesized that snout tentacle secretions are used to provide nutrients to fish larvae when they are under paternal care.     

Effect of tunicamycin on glycogen accumulation in the stratum intermedium and odontoblasts of rat incisor

Summary Repeated injection of rats with tunicamycin over two days induced a 1- to 5-fold increase in glycogen. This accumulation occurred in the stratum intermedium of the enamel organ and in young secretory odontoblasts. In rats injected over 3 days, the number of glycogen particles was at least 10 times larger than in control rats, and large glycogen accumulations were observed in the cytosol of these two groups of cells. These results were obtained by staining with periodic acid-thiocarbohydrazide and silver proteinate, a specific method for the detection of glycoconjugates containing vic-glycol groups. The existence of a relationship between these local cytosolic accumulations of glycogen and the developmental stage of certain groups of cells was shown by the changes that occurred in glycogen distribution. The present results suggest that the stratum intermedium supplies energy for precursor transport.     

The prevalence of proviral bovine leukemia virus in peripheral blood mononuclear cells at two subclinical stages of infection.

The bovine leukemia virus (BLV) is an oncogenic retrovirus that is associated with the development of persistent lymphocytosis (PL) and lymphoma in cattle. While B lymphocytes have been shown to be the primary cellular target of BLV, recent studies suggest that some T lymphocytes and monocytes may be infected by the virus. Because virally altered functions of monocytes and/or T cells could contribute to the development of lymphoproliferative disease, we sought to clarify the distribution of the BLV provirus in subpopulations of peripheral blood mononuclear cells in seropositive cows with and without PL. CD2+ T cells, monocytes, and CD5+ and CD5- B cells were sorted by flow cytometry and tested for the presence of BLV by single-cell PCR. We did not obtain convincing evidence that peripheral blood monocytes or T lymphocytes contain the BLV provirus in seropositive cows with or without PL. In seropositive cows without PL (n=14), BLV-infected CD5+ and CD5- B cells accounted for 9.2% +/- 19% and 0.1% +/- 1.8% of circulating B lymphocytes, respectively. In cows with PL (n=5), BLV-infected CD5+ and CD5- B cells accounted for 66% +/- 4.8% and 13.9% +/- 6.6% of circulating B lymphocytes, respectively. The increase in lymphocyte numbers in cows with PL was entirely attributable to the 45-fold and 99-fold expansions of infected CD5+ and CD5- B-cell populations, respectively. Our results demonstrate that B cells are the only mononuclear cells in peripheral blood that are significantly infected with BLV. On the basis of the absolute numbers of infected cells in seropositive, hematologically normal animals, there appear to be differences in susceptibility to viral spread in vivo that may be under the genetic control of the host.     

Micronucleus frequency in peripheral blood lymphocytes and exfoliated buccal cells of untreated cancer patients

Some genetic diseases may increase the cellular instability. Since most human tumors have some genetic base, this study was undertaken for the genetic instability in cancer patients by micronucleus analysis, a mutation-screening test, which is more practical and economic technique than metaphase analysis carried out for chromosomal aberrations. Genetic changes were assessed in untreated cancer patients (lung, stomach and colon cancer) by different genotoxical screening methods: the cytokinesis-block micronucleus test and the buccal mucosa cell micronucleus test. The evaluation of micronuclei number in peripheral blood lymphocytes and buccal cells showed genomic instability in somatic cells. There was a significant increase in the number of micronuclei in cancer patients prior to the initiation of chemotherapy, and/or radiotherapy compared with healthy human subjects. Furthermore, there was no significant difference between smokers and non-smoking groups or male and female groups. These results suggest that cancer in humans is characterized by an increase of chromosomal damage and thus, the micronucleus assay carried out here may be useful in routine cytogenetic studies of cancer. The text was submitted by the authors in English.     

Cytofluorimetric research on the glycogen content of the hepatocytes in patients with various forms of alcoholic liver lesions

By cytofluorometry employing the cytofluorometric PAS reaction, a study was made of the total glycogen and of its two fractions in liver parenchymal cells, both in the norm and in patients with chronic alcoholism (alcoholic steatosis, chronic alcoholic hepatitis, and mixed forms of alcoholic-viral hepatitis, viral hepatitis with steatosis and also viral hepatitis). The examination was performed on preparations-smears of isolated hepatocytes, obtained from the live puncture liver biopsies. The quantitative analysis has shown the increase in the total glycogen content in hepatocytes of patients with alcoholic hepatitis in comparison with the norm and with chronic viral hepatitis. The transition from a reverse stage--alcoholic steatosis--to alcoholic hepatitis was accompanied by a sharp increase in the total glycogen content and by an obvious change in the ratio of glycogen fractions, towards the hard soluble fraction in liver cells. The quantitative analysis of glycogen fractions in liver cells of patients with chronic alcoholic disease may be an appreciated marker of differential diagnostics of different stages and forms of alcoholic liver disease.     

Problems in the genetics of bovine leukemia. II. The effect of fathers and mothers on the leukemia disease frequency in the progeny

Geneology of 14,000 animals which are the progeny of 554 bulls are studied, 2060 of them having leucosis. Differences between bulls in the frequency of the disease in daughters are observed. The morbidity of daughters of bulls having leucosis is higher than for the population in the average. The morbidity of the animals depends on the linear relation animals. "Leucosis" families having a high concentration of ill animals for several generations, and families resistant to leucosis are revealed. Daughters of leucosis mothers got ill more often than those of healthy animals. The coefficient of heritability of leucosis ranges from 0.07 to 0.50. Concordancy for leucosis in unisexual twins is 74.1%. Insignificant increase in leucosis is found for the last three generations. Predisposition for leucosis is characterized by a complex hereditary condition. The portion of genetic factors is quite enought to conduct the animal selection for leucosis resistance.     

Glycogen in pancreatic islets of steroid diabetic rats

Summary In the islets of the rat pancreas, steroid diabetes induced by triamcinolon-acetonid leads to degranulation of the B cells and glycogen infiltration. The glycogen cannot be satisfactorily detected using methods like the chromic acid technique according to Bauer, staining with Best's carmine, or the usually applied periodic acid-Schiff (PAS) reaction. Glycogen detection is improved, however, when lead tetraacetate is used in place of periodic acid as oxidizing agent. When combining the carbohydrate detection method with the peroxidase — antiperoxidase (PAP) method used for immunocytochemical detection of the various pancreatic islet hormones, paraffin sections reveal that glycogen is primarily localized in granulated B cells; the degranulated B cells also contain glycogen, though in smaller amounts. In contrast, the islet cells containing somatostatin, glucagon and pancreatic polypeptide are nearly free of glycogen.This study was supported by the Deutsche Forschungsgemeinschaft K1 426/2     

Electrolyte composition of cow and calf tissues in health and in acute digestive disturbances]

Sodium, potassium, calcium, magnesium, manganese, zinc and chloride contents in the blood plasma of calves at parturition have been established to correspond to their levels in blood of cows during their calving period. Iron and inorganic phosphorus contents in calves blood plasma appeared to increase and copper content to be lower in this period as compared to postnatal period. By the third day of postnatal ontogenesis sodium concentration in calves blood decreased, copper level increased and the rest indices of water-salts metabolism in calves and adult animals were alike. Digestion disturbances in the calves were accompanied by changes in levels and magnitude of Na+/K+ ratio, magnesium, iron, copper, manganese and zinc contents in blood, liver and kidneys as well as Ca++/Pi ratio in mitochondria and cytosol of liver and jejunum mucose layer cells in comparison with clinically healthy animals.     

Immune modification of the pathogenesis of Streptococcus uberis mastitis in the dairy cow

Abstract Two groups of 4 cows were vaccinated subcutaneously with live Streptococcus uberis strain 0140J or a surface extract derived from the same strain, at 14 days prior to the cessation of lactation (drying off) and at calving. Both groups also received an intramammary administration of the surface extract 7 days after drying off. A third group of unvaccinated animals acted as controls. Following intramammary challenge of two quarters per cow with the vaccine strain, all quarters on control cows and those vaccinated only with surface extract developed clinical mastitis. However, only 12.5% of challenged quarters on cows which were vaccinated with live bacteria developed clinical mastitis. In addition, the numbers of bacteria in the milk following challenge were 10⁵ times higher from the control and extract vaccinated cows than those which received live vaccine. Serum levels of S. uberis specific IgG₂ were elevated in the animals vaccinated with the live organism when compared to that of either extract-vaccinates or controls, whilst S. uberis specific levels of IgG₁ and IgM were similar in all groups throughout the experiment. Specific antibody levels in milk were unaffected by vaccination. Despite increased levels of IgG₂, no increase in opsonic activity was detected in any serum or milk samples. Peripheral blood lymphocytes from animals vaccinated with live organisms showed a considerable increase in proliferative response to S. uberis antigen in vitro when compared with lymphocytes from control and extract-vaccinated animals. These results suggest that neutrophils and specific opsonising antibody may not form the major defence against infection with S. uberis .     

Cytochemical, immunocytochemical and ultrastructural observations on leukocytes and thrombocytes of fat snook (Centropomus parallelus)

The cytochemical, immunocytochemical and ultrastructural characteristics of leukocytes and thrombocytes in the peripheral blood of the fat snook (Centropomus paralellus) - a fish occurring in Brazil - were investigated. The cytochemical methods were performed to demonstrate four enzymatic reactions - o-toluidine-hydrogen peroxide, naphtol AS-MX phosphate, naphtol AS-BI phosphate and alpha-naphtil acetate to detect myeloperoxidase (MPO), alkaline phosphatase (ALP), acid phosphatase (ACP) and non-specific esterase (α-NAE), respectively - and two non-enzymatic ones - Periodic-Acid Schiff (PAS) and Sudan black B (SBB) to detect the occurrence of glycogen and phospholipids, respectively. Immunocytochemical method utilizing polyclonal rabbit antibody against mammal metalloproteinases (MMPs) 2 and 9 were done. Standard method for Electron Microscopy (EM) was applied for the ultrastructural study. The cytochemical reactions were positive in neutrophils for MPO, ACP, α-NAE, glycogen and phospholipids; in lymphocytes for ACP and α-NAE; in monocytes for ACP and α-NAE and in thrombocytes for ACP, α-NAE and glycogen. Only neutrophils were positive for MMPs 2 and 9, and none of the cells studied were positive for ALP. Ultrastructurally: 1) neutrophil showed a spherical shape with a spherical, indented or lobulated euchromatic nucleus, and cytoplasm containing granules of varied sizes and mitochondria of varied shapes and sizes. The nucleus/cytoplasm relation and the size of granules suggest neutrophil maturation in peripheral blood; 2) lymphocytes showed partially heterochromatic nucleus and minimal cytoplasm; 3) monocytes had long cytoplasmic projections, an indented nucleus, evident nucleolus and cytoplasm with granules of varied sizes and vacuoles; 4) thrombocytes were predominantly elliptical or roughly spherical in shape, had a partially heterochromatic nucleus and cytoplasm containing electron-dense granules, intricate canalicular system and vacuoles occasionally holding phagocytic material.     

Micronucleus frequency in peripheral blood lymphocytes and exfoliated buccal cells of untreated cancer patients

Some genetic diseases may increase the cellular instability. Since most human tumors have some genetic base, this study was undertaken for the genetic instability in cancer patients by micronucleus analysis, a mutation-screening test, which is more practical and economic technique than metaphase analysis carried out for chromosomal aberrations. Genetic changes were assessed in untreated cancer patients (lung, stomach and colon cancer) by different genotoxical screening methods; the cytokinesis-block micronucleus test and the buccal mucosa cell micronucleus test. The evaluation of micronuclei number in peripheral blood lymphocytes and buccal cells showed a genomic instability in somatic cells. There was a significant increase in the number of micronuclei in cancer patients prior to the initiation of chemotherapy, and/or radiotherapy compared with healthy human subjects. Furthermore, there was no significant difference between smokers and non-smoking groups or male and female groups. These results suggest that cancer in humans is characterized by an increase of chromosomal damage and thus, the micronucleus assay carried out here may be useful in routine cytogenetic studies of cancer.     

Electrophoretic mobility of human lymphocytes purified by nylon columns and spontaneous rosettes]

Cell electrophoresis enables the separation of the lymphocytes in normal human blood into two principal groups, as a function of their migration speed in relation to 1 mum.sec -1..v-1.cm. In 42 healthy adults, 19,9 % of the lymphocytes have a slower migration, and 80,1 % a faster migration than the reference speed. Two known methods are used for the selection of the lymphocytic populations : spontaneous rosetting with sheep red blood cells, a property of the T lymphocytes, and the adherence to nylon wool columns, which is dominant in the case of B lymphocytes. The cells which do not form spontaneous rosettes, and the cells adhering to nylon wool columns have above all a slow migration. On the contrary, cells which do not adhere to nylon columns have a fast migration. These arguments are in favour of the T nature of the rapid migrating lymphocytes, and of the B nature of the slow migrating lymphocytes.     

Some comments on the PAS reaction of glycogen

Synopsis The periodic acid-Schiff (PAS) reaction has been studied in sections of mouse liver using a self-assembled microspectrophotometer. Increased colour intensity was obtained up to 4 hr of oxidation with periodic acid and 2 hr treatment with Schiff's reagent. The oxidation curve showed an initial, steep increase in colouration with a levelling off afterwards, that could not be attributed to loss of aldehyde groups. The results obtained from carrying out the PAS reaction on sections pretreated with -amylase suggest that the reaction takes place in two phases in which the outer glucosyl groups of the glycogen molecule are oxidized more rapidly than the inner ones.