Dual-factor Synergistically Activated ESIPT-based Probe: Differential Fluorescence Signals to Simultaneously Detect α-Naphthyl Acetate and Acid α-Naphthyl Acetate Esterase.

α-Naphthyl acetate esterase (α-NAE) and acid α-naphthyl acetate esterase (ANAE), a class of special esterases, are important for lymphocyte typing and immunocompetence-monitoring. As such, the simultaneous detection of α-NAE and ANAE has become a target to effectively improve the accuracy in lymphocyte typing. Therefore, we developed a dual-factor synergistically activated ESIPT-based probe (HBT-NA) to detect α-NAE and ANAE sensitively, rapidly, and simultaneously in a differential manner. HBT-NA exhibits differential fluorescence signal outputs toward small changes of α-NAE and ANAE activities. HBT-NA displays a weak fluorescence signal at 392 nm over a pH range from 6.0 to 7.4. However, when it interacts with α-NAE (0-25 U) at pH = 7.4, the fluorescence intensity at 392 nm enhanced linearly within 60 s (F392 nm/F0392 nm = 0.042 Cα-NAE + 1.1, R2 = 0.99). Furthermore, HBT-NA emits ratiometric fluorescence signals (F505 nm/F392 nm) for ANAE (0-25 U) at pH = 6.0 within 2.0 min, exhibiting a good linear relationship (F505 nm/F392 nm = 0.83CANAE - 1.75, R2 = 0.99). The differential fluorescence signals can be used to simultaneously detect the activities of α-NAE and ANAE in solutions and complex living organisms. More importantly, based on the differential fluorescence signals toward α-NAE and ANAE, T lymphocytes and B lymphocytes could be successfully typed and differentiated among nontyped lymphocytes, facilitating the real-time evaluation of their immune functions using flow cytometry. Hence, HBT-NA could be used for the ultrasensitive detection of the enzyme activities of α-NAE and ANAE, the real-time precise typing of lymphocytes, and the monitoring of immunocompetence.

Benzoate mediates the simultaneous repression of anaerobic 4-methylbenzoate and succinate utilization in Magnetospirillum sp. strain pMbN1.

BACKGROUND: At high concentrations of organic substrates, microbial utilization of preferred substrates (i.e., supporting fast growth) often results in diauxic growth with hierarchical substrate depletion. Unlike the carbon catabolite repression-mediated discriminative utilization of carbohydrates, the substrate preferences of non-carbohydrate-utilizing bacteria for environmentally relevant compound classes (e.g., aliphatic or aromatic acids) are rarely investigated. The denitrifying alphaproteobacterium Magnetospirillum sp. strain pMbN1 anaerobically degrades a wide variety of aliphatic and aromatic compounds and is unique for anaerobic degradation of 4-methylbenzoate. The latter proceeds via a distinct reaction sequence analogous to the central anaerobic benzoyl-CoA pathway to intermediates of central metabolism. Considering the presence of these two different anaerobic "aromatic ring degrading" pathways, substrate preferences of Magnetospirillum sp. strain pMbN1 were investigated. Anaerobic growth and substrate consumption were monitored in binary and ternary mixtures of 4-methylbenzoate, benzoate and succinate, in conjuction with time-resolved abundance profiling of selected transcripts and/or proteins related to substrate uptake and catabolism. RESULTS: Diauxic growth with benzoate preference was observed for binary and ternary substrate mixtures containing 4-methylbenzoate and succinate (despite adaptation of Magnetospirillum sp. strain pMbN1 to one of the latter two substrates). On the contrary, 4-methylbenzoate and succinate were utilized simultaneously from a binary mixture, as well as after benzoate depletion from the ternary mixture. Apparently, simultaneous repression of 4-methylbenzoate and succinate utilization from the ternary substrate mixture resulted from (i) inhibition of 4-methylbenzoate uptake, and (ii) combined inhibition of succinate uptake (via the two transporters DctPQM and DctA) and succinate conversion to acetyl-CoA (via pyruvate dehydrogenase). The benzoate-mediated repression of C4-dicarboxylate utilization in Magnetospirillum sp. strain pMbN1 differs from that recently described for "Aromatoleum aromaticum" EbN1 (involving only DctPQM). CONCLUSIONS: The preferential or simultaneous utilization of benzoate and other aromatic acids from mixtures with aliphatic acids may represent a more common nutritional behavior among (anaerobic) degradation specialist than previously thought. Preference of Magnetospirillum sp. strain pMbN1 for benzoate from mixtures with 4-methylbenzoate, and thus temporal separation of the benzoyl-CoA (first) and 4-methylbenzoyl-CoA (second) pathway, may reflect a catabolic tuning towards metabolic efficiency and the markedly broader range of aromatic substrates feeding into the central anaerobic benzoyl-CoA pathway.

Histological and histochemical characteristics of the developing chicken (Gallus gallus domesticus) cecum.

Avian ceca play an important role in liquid absorption, cellulose digestion, and defensive mechanism. This study aims to demonstrate histological and histochemical characteristics of developing chicken cecum. For this purpose, 10 embryos on the 18th day of incubation, 10 chicks on hatching day and 10 chicks on the seventh day post-hatching were used. The histological sections prepared from proximal, middle, and distal parts of cecum were stained with Crossmon's triple stain, periodic-acid Schiff, Alcian blue (pH 2.5), Masson-Fontana's argentaffin silver stain and Gordon and Sweets's silver stain. Alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (ACP-ase) were also demonstrated in the sections. In the proximal part, although the villi were rudimentary on the 18th day of incubation, well-developed villi were seen at seventh day post-hatching. In middle and distal parts, while it was seen that rudimentary folds appeared on the 18th day of incubation, mucosal folds were prominent and short villi were formed on the hatching day and seventh day post-hatching. Goblet cells and enteroendocrine cells were detected from the 18th day of incubation. The lymphoid follicles supported with reticular fibres were seen on the seventh day post-hatching in proximal cecum wall. While ACP-ase (+) lymphocytes were rarely observed, more ANAE (+) lymphocytes were in lymphoid follicles. As a result, development of cecum in chickens has been demonstrated by histological techniques in this study.

A specific esterase and pH logically regulate ESIPT: different kinds of granulocyte sorting.

Simultaneously detecting naphthol AS-D chloroacetate esterase (NAS-DCE) and pH is an effective way to separate different granulocytes, which is of great significance for the analysis of blood. A series of fluorescent small molecules (HBT-ASDs) were designed, whose ESIPT process could be logically regulated by NAS-DCE and pH. One typical molecule, HBT-ASD-2, emits three kinds of fluorescence output signal at 438 nm and 545 nm for NAS-DCE under different pH values (5.0, 7.4 and 10, respectively). According to such differential signals, the acid, neutrophil and alkaline granulocytes can be sorted, and the activity of NAS-DCE can also be simultaneously monitored in real-time. Thus, a simple analytical tool for clinical blood monitoring and analysis is provided.

The determination of the effect of in ovo administered monosodium glutamate on the embryonic development of thymus and bursa of Fabricius and percentages of alpha-naphthyl acetate esterase positive lymphocyte in chicken.

Monosodium glutamate (MSG) is a flavor enhancer commonly used in modern nutrition. In this study, it was aimed to determine the effect of in ovo administered MSG on the embryonic development of thymus, bursa of Fabricius, and percentages of alpha-naphthyl acetate esterase (ANAE) positive lymphocyte by using histological, histometrical, and enzyme histochemical methods in chickens. For this purpose, 410 fertile eggs were used. The eggs were then divided into five groups: group 1 (control group, n = 40 eggs), group 2 (distilled water-injected group, n = 62 eggs), group 3 (0.12 mg/g egg MSG-injected group, n = 80 eggs), group 4 (0.6 mg/g egg MSG-injected group, n = 90 eggs), and group 5 (1.2 mg/g egg MSG-injected group, n = 138 eggs), and injections were performed via the egg yolk. On the 18th and 21st days of the incubation, the eggs were randomly opened from each group until six live embryos were obtained. The embryos of each group were sacrificed by decapitation, and blood, thymus, and bursa of Fabricius tissue samples were taken from the obtained embryos. The MSG-treated groups were found to be retarded embryonic development of thymus and bursa of Fabricius tissue compared to the control and distilled water groups. MSG treatment also resulted in reduced lymphoid follicles count and follicle diameters in bursa of Fabricius (P < 0.05). The percentage of peripheral blood ANAE positive lymphocytes was significantly lower in the MSG-treated groups than in the control and distilled water groups (P < 0.05). In conclusion, it has been found that in ovo administered MSG can adversely affect the embryonic development of thymus and bursa of Fabricius and decrease percentage of ANAE positive lymphocyte.

Morphology and cytochemical patterns of peripheral blood cells of tiger frog (Rana rugulosa).

Background: Tiger frog (Rana rugulosa) is a national second-class protected amphibian species in China with an important ecological and economic value. In recent years, due to excessive human hunting, pollution and habitat loss, the wild population of tiger frog has declined sharply. To protect wildlife resources, the artificial breeding of tiger frogs has rapidly developed in China. Diseases are increasing and spreading among tiger frogs due to the increasing scale of artificial farming. The blood examination is the most straightforward and less invasive technique to evaluate the animal health condition. Thus, it is essential to obtain the normal hematological indicators of tiger frogs. The objective of this study was to investigate the morphometry, microstructure and cytochemical patterns of peripheral blood cells in tiger frogs. Methods: The number of blood cells in tiger frogs was counted on a blood count board, and the cell sizes were measured by a micrometer under light microscope. The morphology and classification of blood cells were studied by Wright-Giemsa staining, and the cytochemical pateerns was investigated by various cytochemical staining including periodic acid-Schiff (PAS), Sudan black B (SBB), peroxidase (POX), alkaline phosphatase (AKP), acid phosphatase (ACP), chloroacetic acid AS-D naphthol esterase (CAE) and α-naphthol acetate esterase (ANAE) staining. Results: Besides erythrocytes and thrombocytes, five types of leukocytes were identified in tiger frogs: neutrophils, eosinophils, basophils, lymphocytes and monocytes. The mean erythrocyte, leukocyte and thrombocyte counts were 1.33 ± 0.15 million/mm3, 3.73 ± 0.04 × 104/mm3 and 1.7 ± 0.01 × 104/mm3, respectively. Small lymphocytes were the most abundant leukocytes, followed by large lymphocytes, Neutrophils, eosinophils and monocytes, basophils were the fewest. Eosinophils were strongly positive for PAS, positive for SBB, POX, ACP, CAE, ANAE, while weakly positive for AKP staining; basophils were strongly positive for PAS, ACP, positive for SBB, CAE, weakly positive for ANAE, negative for AKP, POX staining; neutrophils were strongly positive for ACP, SBB, positive for PAS, POX, weakly positive for AKP, CAE and ANAE staining; monocytes were positive for PAS, SBB, ANAE, weakly positive for ACP, AKP, POX, CAE staining; large lymphocytes and thrombocytes were positive for PAS, ACP, weakly positive for ANAE, while negative for SBB, POX, AKP, CAE; small lymphocytes were similar to large lymphocytes, except for strongly positive for PAS and ACP staining. Conclusions: The blood cell types and morphology of tiger frogs were generally similar to those of other amphibians, while their cytochemical patterns had some notable species specificity.Our study could enrich the knowledge of peripheral blood cell morphology and cytochemistry in amphibians, and provide baseline data for health condition evaluation and disease diagnosis of tiger frogs.

A study of peripheral blood in hedgehogs in Turkey.

The aim of this study was to determine diameters of blood cells, differential counts of peripheral blood leukocytes, alpha-naphthyl acetate esterase (ANAE), acid phosphatase (ACP-ase) activity of some leukocyte types, and enzymatic positivity percentages of peripheral blood lymphocytes in two hedgehogs species, Hemiechinus auritus, the long-eared hedgehog, and Erinaceus concolor, the southern white-breasted hedgehog. Air-dried peripheral blood smears were stained with May-Grünwald-Giemsa stain. ANAE and ACP-ase were stained in glutaraldehyde-acetone-fixed smears. ANAE-positive lymphocytes displayed a dot-like positivity pattern characterized with 1-5 reddish brown cytoplasmic granules, whereas ACP-ase positive lymphocytes displayed a dot-like positivity pattern characterized with 1-3 pinkish cytoplasmic granules. Monocytes gave a diffuse and strong reaction while neutrophils displayed a weak positive reaction for ANAE and ACP-ase. No difference was observed in mean diameters of peripheral blood cells of these species. It was found that lymphocytes made up the majority (64.3% and 65.5%) of leukocytes, followed by neutrophils (23.9% and 23.3%), eosinophils (9.0% and 7.6%), monocytes (1.8% and 2.3%), and basophils (1.0% and 1.3%) in H. auritus and E. concolor, respectively. Mean ANAE positivity oflymphocytes was 36.6% and 51.3% and ACP-ase positivity was 32.1% and 37.5% for H. auritus and E. concolor, respectively. The ANAE positivity of lymphocytes in E. concolor was significantly (P < 0.05) higher than that of H. auritus.

Acute myelomonocytic leukemia negative for alpha-naphthyl acetate esterase stain in a Holstein cow.

A 4-year, 7-month-old Holstein cow presented with anorexia. Physical examination revealed masses in the interscapular region and vagina. Blast cells were detected in the masses and peripheral blood by fine needle aspiration cytology and hematological examination. By bone marrow aspiration, blast cells constituted up to 24.2% of all nucleated cells, and 22% and 2% of non-erythroid cells stained positive for myeloperoxidase and alpha-naphthyl acetate esterase (ANAE), respectively. Pathological examination revealed the mass lesions consisted of a proliferation of tumor cells, which were positive for monocytic markers (HLA-DR and Iba-1). The cow was diagnosed with acute myelomonocytic leukemia (AMML). Even when tumor cells are ANAE-negative, AMML cannot be completely ruled out and should be considered when diagnosing cattle with leukemia/lymphoma.

Characterization of hematopoietic potential of mesenchymal stem cells.

Mesenchymal and hematopoietic tissues are important reservoirs of adult stem cells. The potential of tissue resident mesenchymal stem cells (MSCs) to differentiate into cells of mesodermal and ectodermal lineages has been reported previously. We examined the hypothesis that adherent adipose tissue resident mesenchymal stem cells (ASCs) are capable of generating cells with hematopoietic characteristics. When cultured in differentiation media, clonally isolated ASCs develop into cells with hematopoietic attributes. The hematopoietic differentiated cells (HD) express early hematopoietic (c-kit, PROM1, CD4) as well as monocyte/macrophage markers (CCR5, CD68, MRC1, CD11b, CSF1R). Additionally, HD cells display functional characteristics of monocyte/macrophages such as phagocytosis and enzymatic activity of α-Naphthyl Acetate Esterase. HD cells are also responsive to stimulation by IL-4 and LPS as shown by increased CD14 and HLA-DRB1 expressions and release of IL-2, IL10, and TNF. Taken together, this study characterizes the potential of ASCs to generate functional macrophages in vitro, and therefore paves way for their possible use in cell therapy applications.

Characterization of rabbit stromal fibroblasts derived from red and yellow bone marrow.

Rabbit stromal fibroblasts subcultured from red and yellow bone marrow and implanted beneath the renal capsule form ossicles the hemic cellularity of which mirrors the cellularity of the marrow used for culture. Although the cultured red and yellow marrow cells are similar in fine-structural appearance, they differ strikingly in enzymatic content of alpha-naphthylbutyrate esterase, which is abundant only in the cells derived from yellow marrow. Other observers (20, 21) have proposed that stromal fibroblasts are preadipocytes, and this data suggests that those derived from yellow marrow have the phenotype of more differentiated adipocytes. On the other hand, fibroblasts derived from red and yellow bone marrow show no differences in their profiles of procollagen synthesis. Both types of fibroblasts secrete type III procollagen as the major species, with a I/III ratio of 1:3; in contrast, rabbit dermal fibroblasts have a prominent peak of type I procollagen. The similarity of stromal cells derived from red and yellow bone marrow in procollagen synthesis suggests that the collagen part of the extracellular matrix is not the only basis for their intrinsic difference in capacity for hematopoiesis.

Tissue culture studies in Hodgkin's disease: Morphologic, cytogenetic, cell surface, and enzymatic properties of cultures derived from splenic tumors.

Monolayer and suspension cell cultures prepared from Hodgkin's disease tumors in the spleen were examined microscopically and by cytogenetics, tested for lymphocyte and monocyte cell surface properties, and assayed for enzymes by histochemical and spectrophotometric techniques. Hodgkin's disease monolayer cultures were composed of rapidly proliferating round and polygonal cells that were capable of propagation in vitro for an indefinite period of time. Abnormal aneuploid chromosomes were found in short-term Hodgkin's disease monolayers that had been passaged 16-20 times, and in established cell lines carried in culture longer than 3 yr and passaged more than 200 times. Cells fromHodgkin's disease monolayers contained lysozyme (muramidase), fluoride-resistant alpha naphthol acetate esterase, acid and alkaline phosphatase, and chymotrypsin-like activity. The monolayers did not exhibit specific cell surface markers or phagocytosis. Suspension cultures derived from Hodgkin's disease monolayers were composed of cells with aneuploid karyotypes and similar enzymes. The Hodgkin's disease suspension culture cells had surface receptors for complement and IgGFc, lacked surface or cytoplasmic immunoglobulin, and did not form Erosettes, react with an antithymocyte serum, nor exhibit phagocytosis. Normal monolayer culture cells, derived from adult spleen and human fetal spleen and thymus, were composed of spindle cells with a diploid number of chromosomes that could be carried for only a finite period of time in vitro. Normal cultured cells contained similar esterases and phosphatases, but were devoid of lysozyme and chymotrypsin-like activity. The morphologic, cytogenetic, cell surface, and enzymatic findings indicate that our Hodgkin's disease monolayer and suspension cultures are composed of cells with many properties suggesting an origin from monocytes (macrophages) rather than lymphocytes or fibroblasts. The presence of aneuploid karyotypes is consistent with a neoplastic origin and derivation from a malignant cell of Hodgkin's disease.

Cloned mouse cells with natural killer function and cloned suppressor T cells express ultrastructural and biochemical features not shared by cloned inducer T cells.

We have examined the morphology, cytochemistry, and biochemistry of mouse leukocyte subsets by analyzing cloned leukocyte populations specialized to perform different immunologic functions. Cloned cells expressing high-affinity plasma membrane receptors for IgE and mediating natural killer (NK) lysis and cloned antigen-specific suppressor T cells contained prominent osmiophilic cytoplasmic granules similar by ultrastructure to those of mouse basophils. Both clones also incorporated 35SO4 into granule-associated sulfated glycosaminoglycans, expressed a characteristic ultrastructural pattern of nonspecific esterase activity, incorporated exogenous [3H]5-hydroxytryptamine, and contained cytoplasmic deposits of particulate glycogen. By contrast, cloned inducer T cells lacked cytoplasmic granules and glycogen, incorporated neither 35SO4 nor [3H]5-hydroxytryptamine, and differed from the other clones in pattern of nonspecific esterase activity. These findings establish that certain cloned cells with NK activity and cloned suppressor T cells express morphologic and biochemical characteristics heretofore associated with basophilic granulocytes. However, these clones differ in surface glycoprotein expression and immunologic function, and the full extent of the similarities and differences among these populations and basophils remains to be determined.

Histochemical and histological evaluations of the effects of high incubation temperature on embryonic development of thymus and bursa of Fabricius in broiler chickens.

1. The effects of experimentally induced heat-stress on the embryonic development of bursa of Fabricius and thymus of the chicken were investigated by means of histological and enzyme histochemical methods. 2. In the experiments, 250 fertile eggs of the Ross 308 broiler strain were divided into two groups. The control eggs were maintained under optimal conditions (378 degrees C and 65 +/- 2% relative humidity, RH) during the whole incubation period. Heat stressed eggs were maintained under normal conditions (378 degrees C and 65 +/- 2% RH) until the 10th d of incubation and then exposed continuously (24 h per d) to high temperature (388 degrees C and 65 +/- 2% RH). Blood and tissue samples were taken from 10 animals of each group at d 13, 15, 18 and 21 of incubation and at d 2, 4 and 7 post-hatch. Tissue samples were processed for enzyme histochemical methods in addition to routine histological techniques. 3. The results revealed that egg temperatures were higher than incubator air temperature. Long-term heat-stress (401-406 degrees C egg temperature) retarded development of thymus and bursa of Fabricius. Peripheral blood ACP-ase and ANAE-positive lymphocyte levels of heat-stressed animals were lower than in the controls. 4. These results give some morphological evidence for immunosuppression induced by high temperature exposure during the embryonic development. Temperature distribution and air circulation in incubator should be questioned in the case of lower broiler flock immunity.

Haematology and leucocyte enzyme cytochemistry of a threatened yellow catfish Horabagrus brachysoma (Gunther 1864).

The haematology and leucocyte enzyme cytochemistry of Horabagrus brachysoma, a threatened freshwater catfish endemic to southern India, was studied using standard methods. Intra-specific variation was found for the haematological parameters, but this did not exceed the range of values observed in other catfishes. The relatively high haemoglobin (Hb) concentration may be indicative of an ability to breathe air and high activity. The erythrocytes are fully packed with Hb, revealing the bottom dwelling habit and primitive nature of this catfish. The leucocyte enzyme pattern also showed some variations from those of other fishes. Lymphocytes were positive only for peroxidase (PER) enzyme activity and negative for alkaline phosphatase (LAP), alpha-naphthyl acetate esterase (ANAE) and naphthol ASD chloroacetate esterase (ASDE). Monocytes were weakly positive for ANAE activity and negative for the other three enzymes tested. Neutrophils were negative for LAP, ANAE and ASDE but showed a moderately strong positive reaction for PER. Basophils and eosinophils were found to be devoid of all of these enzymes. Thrombocytes were observed to have weakly positive PER and ASDE, but there was no demonstrable LAP and ANAE activity. A number of characteristics were identified that distinguish this species from other fishes: (1) lymphocytes of H. brachysoma are actively engaged in both phagocytosis and defence mechanisms, while the monocytes participate in cellular defence mechanisms, primarily phagocytosis; (2) thrombocytes function as a protection barrier as well as carrying out their normal function of haemato plug formation during blood clotting. Results from the haematological and leucocyte cytochemical analyses reveal the haematological make-up and effective immune mechanism of this threatened fish and show it to be highly adaptive in nature. The data may be useful in programmes aiming the effective conservation of this species.

Effects of paclitaxel and resveratrol on blood characteristics in rabbits.

We investigated the adverse effects of paclitaxel (PAC) on blood characteristics including the percentage of alpha naphthyl acetate esterase (ANAE)-positive peripheral blood lymphocytes, white cell count, number of neutrophil nuclear lobe and, erythrocyte diameter, and the protective effects of resveratrol (RES) on these characteristics. Male New Zealand rabbits were assigned to four groups. The control group (C) was given 40 ml normal saline, the PAC group was given 5 mg/kg PAC in 40 ml normal saline, the RES group was given 4 mg/kg RES in 40 ml normal saline, and the PAC + RES group was given 5 mg/kg PAC + 4 mg/kg RES in 40 ml normal saline. All injections were performed intravenously once/week for 8 weeks. PAC caused an increased total percentage of lymphocytes and monocytes, which was associated with neutropenia. The PAC group showed a right shift in the lobulation curve of neutrophil nuclei together with a decreased percentage of ANAE-positive lymphocytes. RES reduced the percentage of ANAE-positive lymphocytes, slightly increased the percentage of neutrophil leukocytes and reduced the total lymphocyte count. Administration of RES combined with PAC prevented, while PAC induced, neutropenia and lymphocytosis, and increased the percentage of ANAE-positive lymphocytes.

Leukocyte transmigration in inflamed liver: A role for endothelial cell-selective adhesion molecule.

BACKGROUND/AIMS: This study was designed to investigate the role of endothelial cell-selective adhesion molecule (ESAM), a recently discovered receptor expressed in endothelial tight junctions and platelets, for leukocyte migration in inflamed liver. METHODS: The role of ESAM for leukocyte migration in the liver was analyzed using ESAM-deficient mice in a model of warm hepatic ischemia-reperfusion (90min/30-360min). RESULTS: As shown by immunostaining, ESAM is expressed in sinusoids as well as in venules and is not upregulated upon I/R. Emigrated leukocytes were quantified in tissue sections. Postischemic neutrophil transmigration was significantly attenuated in ESAM-/- mice after 2h of reperfusion, whereas it was completely restored after 6h. In contrast, T-cell migration did not differ between ESAM+/+ and ESAM-/- mice. Using intravital microscopy, we demonstrate that ESAM deficiency attenuates I/R-induced vascular leakage after 30min of reperfusion. The I/R-induced elevation in AST/ALT activity, the sinusoidal perfusion failure, and the number of TUNEL-positive hepatocytes were comparable between ESAM+/+ and ESAM-/- mice. CONCLUSIONS: ESAM is expressed in the postischemic liver and mediates neutrophil but not T-cell transmigration during early reperfusion. ESAM deficiency attenuates I/R-induced vascular leakage and does not affect leukocyte adherence. Despite the effect on neutrophil migration, ESAM-deficiency does not protect from I/R-induced injury.

Effects of long term oral acrylamide administration on alpha naphthyl acetate esterase and acid phosphatase activities in the peripheral blood lymphocytes of rats.

Acrylamide is an important industrial chemical; it also is formed in starch-rich foodstuffs during baking, frying and roasting. Most acrylamide exposure occurs by ingestion of processed foods. We investigated possible immunotoxic effects of extended administration of low doses of acrylamide in rats. To do this, we measured alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (ACP-ase) activities in peripheral blood lymphocytes. Male and female weanling Wistar rats were administered 2 or 5 mg acrylamide/kg/day in drinking water for 90 days. Peripheral blood was sampled at the end of the administration period. We found ANAE staining in eosinophils and T-lymphocytes, but not in monocytes, platelets, B-lymphocytes and neutrophils. ACP-ase was found in B-lymphocytes. We found a significant reduction of the ratio of ANAE:ACP-ase in lymphocytes of the experimental animals compared to controls. We found no statistically significant differences between the doses or sexes. We found that acrylamide ingested in processed foods might affect the immune system adversely by decreasing the population of mature T- and B-lymphocytes.

Cytochemical characterization of peripheral blood cell populations of two Cyprinidae, Carassius auratus and Ctenopharyngodon idellus.

Fish are the most diverse species of all vertebrate groups, and their blood cells have shown variable characteristics in terms of morphology. Cytochemical staining for enzyme activity in blood leukocytes will help assess the immune function of fish. We characterize blood cells from crucian carp (Carassius auratus) and grass carp (Ctenopharyngodon idellus) by using a Diff-Quick stain as well as different cytochemical methods. Blood specimens obtained from crucian carp and grass carp were evaluated after cytochemical staining for acid phosphatase (ACP), alkaline phosphatase (ALP), naphthol AS chloroacetate esterase (AS-DNCE), naphthyl acetate esterase (NAE), α-naphthyl butyrate esterase (NBE), peroxidase (MPO) and periodic acid-Schiff's reaction (PAS) using commercial kits. Blood cell types were evaluated based on their morphological characteristics and the presence or absence of specific chromogen. The expression pattern of enzymes was similar between the two Cyprinidae and was also broadly consistent with other fish species. However, there were some interesting differences detected between crucian carp and grass carp, including naphthol AS chloroacetate esterase activity in monocytes, peroxidase activity and location in thrombocytes. The ACP, ALP and MPO expressions of different leukocytes of the two Cyprinidae were evaluated by Image Pro Plus and were analysed for statistical significant differences. This investigation provides basic haematology and enzyme activity analyses for crucian carp and grass carp and serves as an approach to evaluating the immune response of fish.

Regulatory effects of dermal papillary pluripotent stem cells on polarization of macrophages from M1 to M2 phenotype in vitro.

The M1:M2 macrophage ratio is important for spinal cord injury (SCI) repair. Bone marrow mesenchymal stem cells (BMSCs) can alter macrophage activation, promoting M1 to M2 macrophage conversion and SCI repair; however, clinical BMSC applications have limitations. Previously, we found DPCs to be superior to BMSCs in promoting tissue repair after SCI, which we hypothesized to be mediated by M1 to M2 macrophage conversion. We investigated the regulatory effect of DPCs on M1/M2 macrophage polarization. Dermal papilla cells (DPCs) were isolated from rat vibrissae and characterized. Bone marrow-derived macrophages (BMDMs) were isolated and identified based on specific marker expression, and stimulated to differentiate into M1 macrophages with GM-CSF, IFN-γ, and LPS. These cells were co-cultured with DPCs to evaluate the effect on macrophage differentiation. DPCs expressed dermal papillae-specific markers, including ALP and Sox2, had MSC-expression patterns like those of BMSCs, and were capable of multi-differentiation. BMDMs expressed ANAE and CD68. Three days after induction, differentiated cells exhibited morphology typical of M1-like macrophages and expressed the macrophage marker CD68 and the M1 macrophage markers iNOS, but lacked expression of the M2 macrophage marker CD206. Co-culture with DPCs resulted in a shift to anti-inflammatory M2-like macrophage differentiation, characterized by morphological changes typical of M2 macrophages, downregulation of the characteristic cytokine TNF-α and the proportion of iNOS+ cells, and upregulation of the characteristic cytokine IL-10 and the cell-surface marker CD206. The number of CD206-expressing M2 macrophages also increased. These findings demonstrate that DPCs reprogram macrophages to an anti-inflammatory M2 phenotype, which could improve adverse inflammatory microenvironments and promote tissue repair. Thus, DPCs may be an interesting alternative cell source and merit further investigation in applications for SCI therapy.

Protective effect of quercetin on some hematological parameters in rats exposed to cadmium.

We investigated the effects of quercetin (Q) on some hematological parameters and determined the percentage of alpha-naphthyl acetate esterase (ANAE) positive lymphocytes in rats that had been exposed to cadmium (Cd). Thirty male Wistar albino rats were divided into four groups: control (C), quercetin (Q), cadmium (Cd) and Q + Cd (CdQ). Blood samples were taken to assess erythrocytes (RBC), leukocytes (WBC), hemoglobin levels (Hb), hematocrit values (Hct), platelets (PLT), alpha-naphthyl acetate esterase (ANAE) positive lymphocytes. RBC, Hb, Hct; the number of PLT significantly decreased in the Cd group. To the contrary, these parameters were increased significantly in the CdQ group compared to the Cd group. Although we found a significant increase in total WBC count and neutrophil percentage, the number of lymphocytes decreased in the Cd group compared to the other three groups. Also, the percentage of peripheral blood ANAE positive lymphocytes decreased significantly in the Cd group (p < 0.05). Q exhibits positive effects on some hematological characteristics and the percentage of ANAE positive lymphocyte in cases of acute CD toxicity.