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.

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.

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.

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.

Morphological and cytochemical studies of peripheral blood cells of Schizothorax prenanti.

The morphological and cytochemical studies of peripheral blood cells of Schizothorax prenanti were studied by light and electron microscopy. Erythrocytes, thrombocytes and three types of leucocytes, lymphocytes, neutrophils and monocytes, were distinguished and characterized. In addition to mature erythrocytes, immature and dividing erythrocytes were observed. A few organelles such as mitochondria were distributed in the cytoplasm of erythrocytes. Lymphocytes with heavily clumped heterochromatic nucleus and minimal cytoplasm were classified into small and large lymphocytes. Three different populations of granules, with distinctive ultrastructural aspect, were observed in neutrophils. Monocytes were the fewest leucocytes possessing rich organelles, phagocytized materials and vacuoles. Thrombocytes with various types were the most abundant blood cells among leucocytes and contained a prominent nucleus with dense bands of heterochromatin and many cytoplasmic vacuoles. Periodic acid-Schiff staining was positive in neutrophils, monocytes, lymphocytes and thrombocytes, but not in erythrocytes. Peroxidase-positive staining was observed in neutrophils and monocytes, but not in erythrocytes, lymphocytes and thrombocytes. Only neutrophils were positive for oil red O. Except for erythrocytes, the other blood cells stained positively for acid phosphatase. Only neutrophils and monocytes were positive for α-naphthyl acetate esterase. None of the cells studied were positive for alkaline phosphatase. The morphologic and cytochemical features of blood cells of S. prenanti are similar to those of other fish. This investigation may be helpful as a tool to monitor the health status of cultured S. prenanti and will grant early detection of clinical pathology.

NSE/αNAE positivity in B-lineage acute lymphoblastic leukemia: revisiting a potential cytochemical diagnostic pitfall.

Cytochemical staining for leukemia typing is declining in hematology laboratories, but the use of flow cytometry may not be possible in some settings. Aberrant cytochemical nonspecific esterase/α-naphthyl acetate esterase (NSE/αNAE) positive B-lymphoblasts can cause confusion with monoblasts, a potentially dangerous pitfall. This unusual cytochemical NSE/αNAE positivity had been associated with relatively poorer outcome of acute lymphoblastic leukemia (ALL) in the era prior to the advent of routine multicolor flow cytometric immunophenotyping. We reviewed morphological, cytochemical and flow-cytometric data from five cases of B-lineage ALL that showed NSE/αNAE positivity and were diagnosed definitively using multi-parametric flow cytometric immunophenotypic analysis. Diffuse or dot-like (localized) strong cytochemical NSE/αNAE activity was detected in all cases and all showed one or more features of high risk disease. The number of NSE/αNAE positive blasts in the marrow varied from 10 to 75%. The morphological differential diagnoses included T-lymphoid lineage ALL and acute monoblastic leukemia (AML-M5). Flow cytometric data revealed B-lineage antigens and the absence of monocytic or other myeloid markers resolved the diagnosis. These cases underscore the importance of immunophenotyping in all cases of suspected ALL regardless of the cytochemical findings. Although the numbers are small, the association with high risk disease observed in all five of our cases may corroborate the previously reported poor prognostic value of such aberrant cytochemical staining.

Histochemical detection of platelet esterase activity in the bone marrow postmortem: can megakaryocytes serve as indicators for time since death?

AIMS: α-Naphthyl acetate esterase (ANAE) is one of the few enzymes that are histochemically detectable on formalin-fixed paraffin-embedded tissue. In bone marrow (BM) biopsies, ANAE staining highlights megakaryocytes. We investigated autopsy BM to determine whether ANAE staining intensity (SI) was associated with postmortem intervals (PMI, period between death and autopsy), and thus could allow the time of death of a patient to be deduced. METHODS: ANAE-stained BM slides of 74 forensic and pathology autopsies as well as 22 biopsies were histologically evaluated and their SIs semiquantitatively graded. RESULTS: ANAE-SIs did not differ between men and women and slightly decreased with age. Biopsies had significantly higher ANAE-SIs than pathology cases. In autopsies, ANAE-SIs were not associated with PMI, except for cases with PMI ≥7 days which were consistently ANAE-negative. CONCLUSIONS: ANAE-SIs in postmortem BM samples were independent of PMI. Thus, ANAE staining of BM megakaryocytes cannot serve as an indicator for time-since-death of a patient.

The effect of acrylamide on alpha-naphthyl acetate esterase enzyme in blood circulating lymphocytes and gut associated lymphoid tissues in rats.

The aim of this study is to determine the functional effects of the acrylamide (AA) administrated by oral gavage on the peripheral blood lymphocytes (PBL) and the Gut Associated Lymphoid Tissue (GALT) in male Sprague-Dawley rats using alpha-naphthyl acetate esterase (ANAE) demonstration. For this purpose, two separate experiments were performed with Sprague Dawley rats. In Experiment-I rats were gavaged with 0, 30, 45 and 60 mg/kgb.w. AA for five consecutive days and in Experiment-II rats were gavaged with 0, 125, 150, and 175 mg/kg/b.w. AA for single oral dose. Animals were sacrificed 24 h after the last treatments in both experiments by servical dislocations under ether anaesthesia. Blood samples were collected from the heart in heparinized (10 UI heparin/ml(-1) of the blood) tubes before sacrification and lymphoid tissue samples from the ileal Peyer's patches (IPPs) were taken and processed for histochemical demonstration of ANAE following the sacrification. The lymphoid follicles of the IPPs of animals given 125, 150 and 175 mg/kgb.w. AA were markedly reduced in size. Germinal centres (GCs) markedly regressed in AA-treated animals compared with those of controls. ANAE-positive lymphocyte depletion of IPPs was very prominent in the high doses AA-treated animals. In the animals treated with 30, 45, and 60 mg/kg b.w. AA, the IPPs had similar histology to those of the controls. ANAE-positive peripheral blood lymphocyte levels significantly decreased in AA exposed groups in a dose dependent manner (p<0.05). In conclusion, AA has detrimental effects on peripheral blood lymphocytes (PBL) and the Gut Associated Lymphoid Tissue (GALT) in rats.

Hematology and enzyme histochemistry of peripheral blood lymphocytes in domestic pigeon (Columba livia f. domestica).

The purpose of our study was to determine the percentages of alpha-naphthyl acetate esterase (ANAE)-positive and acid phosphatase (ACP)-positive peripheral blood lymphocytes (PBL) in both sexes of one-year-old domestic pigeons (Columba livia f. domestica). Blood samples obtained from 12 healthy domestic pigeons were used. The mean percentages of ANAE-positive PBL for females and males were 47.8% and 48.8%, respectively, whereas the mean percentages of ACP-positive PBL were 67.5% and 68.6%, respectively. The proportions of PBL were 49.3% and 48.6% in males and females, respectively.

Histological and enzyme histochemical investigation of the hemal nodes of the hair goat.

We investigated the structure of the hemal node in six healthy hair goats using histological and enzyme histochemical methods. After processing, tissue sections were stained with Crossman's trichrome, Gordon-Sweet's silver and Pappenheim's panoptic stains. Alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (ACP-ase) were demonstrated in frozen sections. Hemal nodes were encapsulated by connective tissue and few smooth muscle cells. Several trabeculae originated from the capsule and extended into the hemal node. A subcapsular sinus was present beneath the capsule and was continuous with the deeper sinuses. Subcapsular and deep sinuses were filled with erythrocytes. The parenchyma consisted of lymphoid follicles, diffuse interfollicular lymphocytes and irregular wide lymphoid cords. Cortical and medullary regions were not distinct. ANAE (+) and ACP-ase (+) cells were located mainly in the germinal centers of the lymphoid follicles and also were scattered equally in the interfollicular region and lymphoid cords. Monocytes, macrophages and reticular cells displayed a diffuse positive reaction, whereas localized granular positivity was observed in lymphocytes. We demonstrated that the general structure of the hair goat hemal nodes is similar to that of other ruminant species.

Investigation of α-naphthyl acetate esterase and acid phosphatase in the peripheral blood leukocytes of greyhounds.

The purpose of our study was to determine the percentages of α-naphthyl acetate esterase (ANAE)- and acid phosphatase (ACP)-positive peripheral blood lymphocytes (PBL), the presence of the ANAE and ACP enzymes in leukocytes, and the proportion of PBL in greyhounds. Peripheral blood samples were collected from the cephalic antebrachial vein of 14 (7 animals of each sex) healthy 1-2-year-old greyhounds. Mean percentages of ANAE-positive PBL were found to be 73.29 ± 0.95% in female and 74.29 ± 2.21% in male dogs. The difference between mean values of the genders was not statistically significant. The ACP values were 36.00 ± 2.94% for females and 33.57 ± 2.15% for males. No significant differences were found with regard to gender. For both enzymes, although monocytes and eosinophilic granulocytes displayed a positive reaction, neutrophils gave negative reactions. The proportion of PBL was 36.29 ± 5.31% and 33.00 ± 2.38 % in female and male dogs, respectively. The differences were not significant.

Neutrophil infiltration and oxidant-production in human atherosclerotic carotid plaques.

To clarify the clinical implications of neutrophils in vulnerable plaques we evaluated the function and activity of infiltrated neutrophils in an atherosclerotic plaque, focusing on oxidant production. A histopathological investigation was performed using carotid arterial samples obtained from seven patients. The atherosclerotic plaques were examined cytochemically for naphthol-ASD-chloroacetate esterase activity and oxidant-production, and immunohistochemically using N-formyl peptide receptor-like 1 (fPRL1)-, CD66b-, CD68- or p22phox-specific antibodies. The cytoplasmic fPRL1 intensity value of the neutrophils in the plaque was estimated using an activity index. Naphthol-ASD-chloroacetate esterase activity was found in cells located in the atherosclerotic plaque, indicating that the cells were neutrophils. The cytoplasmic fPRL1 intensity value of the neutrophils in the plaque decreased to approximately 60% of the intensity observed in the capillary vessels. Oxidant-production was also detected in the plaques, and both neutrophils and macrophages were observed at the corresponding oxidant-production sites. p22phox expression was also located in the same areas in which oxidant-production was observed in these plaques. We could not directly evaluate how much ROS generated from the infiltrated neutrophils contributed the plaque vulnerability followed by its rupture. However, the infiltrated neutrophils in the atherosclerotic plaques morphologically appeared activated and were actively generating oxidant, implying that neutrophils, together with macrophages, infiltrate into atherosclerotic plaques and contribute to plaque vulnerability.

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.

Mast cell reactivity at the margin of human skin wounds: an early cell marker of wound survival?

Detecting the vitality of mechanical skin wounds (antemortem versus postmortem injury) in human cadavers remains a specifically forensic problem. To determine whether skin mast cells (MCs) are activated during the very early phase of human wound healing we performed a histomorphometric evaluation of the extent of MC enzyme loss as an indication of MC degranulation at the wound margins of skin wounds in 64 human cadavers. We compared the number of tryptase-reactive MCs, which are said not to loose all of their enzyme activity during degranulation process, with the number of naphthol AS-D chloroacetate esterase (NAS-DClAE)-positive MCs, which loose their complete enzyme activity in the form of enzyme-positive granula after activation. The enzyme activity was evaluated on sequential histological sections after autopsy as an indirect quantification of the number of degranulated MCs. Most of the victims had died within 10-60 min after injury (n=50), 12 survived between 60 min and 24h, and only 2 victims survived more than 24h (12 days each). The number of enzyme-positive MCs were counted in six successive visual fields (0.785 mm(2)) on the one hand located parallel to and--on the other hand--at increasing distances outward from the wound margins. In victims surviving the injury less than 60 min the average number of NAS-DClAE-reactive MCs next to the wound margin was significantly lower than the number of tryptase-reactive MCs. The extent of the reduction in NAS-DClAE-reactive MC counts correlated inversely with the distance from the wound edges. Our findings show that MCs undergo very early loss of NAS-DClAE activity at wound margins, and thus appear to be an early cell marker of wound survival. However, definitive evidence that the enzyme loss (degranulation) represents a vital process can only be obtained by comparing MC enzyme loss induced by injury during intact circulation with the MC reaction to injury inflicted very shortly after cardiac arrest, a question that can only be answered by animal experiments.

Determination of acid alpha-naphthyl acetate esterase enzyme activity in peripheral blood leukocytes of gazelles (Gazella subgutturosa).

We examined gazelle peripheral blood leucocytes using the alpha-Naphthyl acetate esterase (ANAE) staining technique (pH 5.8). Our purpose was to determine the percentage of ANAE positive lymphocytes. The proportion of ANAE positive T-lymphocytes was 72%. T-lymphocytes showed an ANAE positive reaction, but eosinophilic granulocytes and monocytes also showed a positive reaction. By contrast, no reaction was detected in B-lymphocytes, neutrophil granulocytes or platelets. The reaction observed in T-lymphocytes was a red-brown coloration, usually 1-2 granules, but enough granules to fill the cytoplasm were detected rarely. As a result of ANAE enzyme staining, we concluded that the staining technique can be used as a cytochemical marker for gazelle T-lymphocytes.

Pisosterol induces monocytic differentiation in HL-60 cells.

The aim of this study was to determine whether the antiproliferative effects observed for pisosterol, a cytotoxic triterpene isolated from Pisolithus tinctorius, are related to cell differentiation induction using HL-60 cell line as a model. Also, the effects of pisosterol on normal human cells were examined in peripheral blood mononuclear cells (PBMC). The effects on cell viability and morphological changes were the first indications showing that pisosterol induces HL-60 differentiation. The demonstration of blue tetrazolium reduction in HL-60 cells exposed to pisosterol demonstrated differentiation in a dose- and time-dependent manner, reaching a maximum effect after 72 h incubation at 5 microg/mL. Assays for alpha-naphthyl acetate esterase activity indicated that pisosterol triggers differentiation towards a monocytic cell-like pathway. The antiproliferative effect of pisosterol was determined by inhibition of DNA synthesis based on BrdU incorporation into HL-60 proliferating cells. It appears that pisosterol-treated cells, despite displaying a differentiated phenotype, continued to proliferate at all doses tested after 72 h, with a slightly decrease at 5 microg/mL. Apoptosis was observed in pisosterol-treated cells in a dose-dependent way. Nevertheless, after the same period of incubation, no cytotoxicity was detected in PBMC in the presence of pisosterol even at 25 microg/mL, providing some evidence that pisosterol may be selective for tumor cells. The mechanisms underlying the effect of pisosterol in leukemia cells indicates the induction of a monocytic cell-like differentiation, suggesting that this compound could be used in the development of new pharmacological tools with potential therapeutic value in the management of leukemia with fewer side effects.

The macrophage chemotactic activity of Streptococcus agalactiae and Streptococcus iniae extracellular products (ECP).

The ability of Streptococcus agalactiae and Streptococcus iniae to attract macrophages of Nile tilapia (Oreochromis niloticus) was investigated. The extracellular products (ECP) from S. agalactiae and S. iniae were tested in vitro for macrophage chemotaxis using blind-well chambers. The macrophages were obtained from the peritoneal cavity 4-5 days after intraperitoneal injection of squalene. Both macrophage chemotactic and chemokinetic activities were demonstrated using the S. agalactiae ECP. However, only chemotactic activity was shown for S. iniae ECP. High-pressure liquid chromatography fractionation revealed that semi-purified S. agalactiae and S. iniae ECPs had estimated molecular weights of 7.54 and 19.2kDa, respectively. The prominent chemotactic activities of ECP from S. agalactiae and S. iniae are likely to be involved in the proinflammatory responses of macrophages to S. agalactiae and S. iniae infections.

Classification of haematopoietic cells and haemocytes in Chinese prawn Fenneropenaeus chinensis.

It is commonly believed that crustacean haemocytes originate from a specialised haematopoietic tissue (HPT), whereas the differentiation relationship between HPT cells and circulating haemocytes is still not clearly understood. The HPT cells and haemocytes of Fenneropenaeus chinensis were characterised using morphological and histochemical methods. Three types of HPT cells were identified under the transmission electron microscope (TEM). Type 1 cells had high N/C ratios, developed dispersed chromatins and no cytoplasmic granules. Type 2 cells had smaller size, developed condensed chromatins and cytoplasmic granules, which were homogeneous or striated in type 2a cells, and homogeneous in type 2b cells. We deduce that type 1 cells may give rise to type 2 cells in terms of the presence of possible intermediates between type 1 and type 2 cells. The circulating haemocytes were divided into three populations, i.e. hyaline haemocytes (HH), small granular haemocytes (SHG) and large granular haemocytes (LGH), based on Wright-Giemsa staining and TEM observation. Comparing the HPT cells with the circulating haemocytes, type 2a cells of HPT may represent the HH due to similar granule types, cell size and N/C ratios, and type 2b cells may be the young and immature LGH. By Wright-Giemsa and acid alpha-naphthyl acetate esterase staining, the intermediates between the HH and SGH were observed, which indicates that the SGH may be derived from the HH in the circulatory system. Therefore, it is suggested that the F. chinensis haemocytes could be divided into two haemocyte lineages, i.e. the HH-SGH and LGH lineage.

Effect of a mixture of iprobenfos and malathion on the development of malathion resistance in the mosquito Culex pipiens pallens Coq.

A malathion-resistant (RM) strain of Culex pipiens pallens Coq was obtained by successively selecting a field population with malathion in the laboratory. The synergistic effect of iprobenfos on malathion toxicity and alpha-naphthyl acetate (alpha-NA) esterase assay revealed that malathion resistance in the RM strain was associated with increased alpha-NA esterase activity and the synergism was mainly due to the inhibition by iprobenfos of this activity. There was no difference in alpha-NA esterase activity between the larvae and female adults in the susceptible (S) strain, but the activity in the adults was 13-fold higher than in the larvae of the RM strain. To understand the effect of the application of a mixture of iprobenfos and malathion on the evolution of malathion resistance, an artificial strain (Syn) was generated by mixing the RM and S strains with 0.1 frequency of the malathion-resistant individuals. The offspring of the Syn strain were divided into two sub-strains, Rm and Rm+ibp, which were successively treated with, respectively, malathion alone and malathion + iprobenfos (1:2) at LC70. In the mixture, the fungicide iprobenfos acted as a synergist of malathion. After treatment for 10 generations, the resistance level to malathion was 317.4-fold for the Rm sub-strain, whereas for the Rm+ibp sub-strain it was only 38.9-fold, compared with the Syn strain. Similar results were obtained by measurement of alpha-NA esterase activity from both larvae and female adults. The alpha-NA esterase activities in larvae and female adults at F10 generation were 2.6- and 10.9-fold from the Rm+ibp sub-strain and 5.7- and 98.5-fold from the Rm sub-strain, respectively, compared with the Syn strain. The above results suggested that iprobenfos, although it cannot completely stop or prevent the onset of malathion resistance, could dramatically delay its evolution.

Relative contributions of enzyme cytochemistry and flow cytometric immunophenotyping to the evaluation of acute myeloid leukemias with a monocytic component and of flow cytometric immunophenotyping to the evaluation of absolute monocytoses.

We evaluated the contributions of enzyme cytochemical stains and flow cytometric immunophenotyping (FCI) data to detection of monocytic cells (MCs) in acute myelomonocytic and acute monocytic leukemias (AMMLs and AMoLs) and compared FCI findings in AMoL, chronic myelomonocytic leukemia (CMML), and normal peripheral blood (PB) and bone marrow (BM) monocytes to classify and evaluate absolute monocytoses (AMs). We reviewed 10 AMMLs and 6 AMoLs with a-naphthyl-acetate esterase (ANAE) and a-naphthyl-butyrate esterase stains and a complete FCI profile and compared FCI data for 6 AMoLs, 7 CMMLs, 2 AMs, and normal monocytes. We confirmed increased sensitivity of ANAE staining to FCI data in detecting MCs in AMML and AMoL. CD14 was insensitive for confirming MCs; other characteristic markers of MCs were absent or partially lost in AMML and AMoL. Aberrant expression of CD56 (detected in 50% of AMMLs and AMoLs), CD34, and CD117 indicated malignancy. The mature MCs of the CMMLs revealed variable FCI abnormalities (partial loss of CD13, CD14, and CD15; expression of CD56), as in the monoblasts of AMoL. These FCI abnormalities in morphologically mature MCs might indicate markers for CMML. AMs revealed FCI abnormalities, indicating clues to their correct classification as CMML.