Bacterial N-formyl Peptides Reduce PMA- and Escherichia coli-Induced Neutrophil Respiratory Burst in Term Neonates and Adults.

Neutrophil migration and respiratory burst are the prerequisite for efficient first line defense against invading microorganisms. However, migration and respiratory burst can be compromised in adults and especially in newborn infants, where sustained neutrophil accumulation, uncontrolled burst and reduced scavenging of ROS might cause inadvertent tissue damage due to uncontrolled inflammation. The aim of this study was to investigate the modulatory effect of the chemoattractants formyl-methionyl-leucyl-phenylalanine (fMLP) and IL-8 on respiratory burst in neutrophils from term newborn infants and adults. Whole blood from the umbilical cord of 17 healthy term newborn infants delivered by caesarean section and from 17 healthy adults as reference was preincubated with fMLP or IL-8 and stimulated with PMA or Escherichia coli bacteria. Respiratory burst was quantified by flow cytometry analysis of dihydrorhodamine 123 fluorescence. fMLP reduced the PMA-induced respiratory burst of neutrophils from newborn infants and adults by 12% and 21%, respectively (P < 0.05). E. coli-induced burst was also reduced by fMLP in neutrophils from newborn infants (10%; P < 0.01) and adults (6%; P < 0.05). No such changes were observed with IL-8. Similar respiratory burst in response to single stimulus with PMA or E. coli was observed in both newborn infants and adults. fMLP reduced PMA- and E. coli-induced respiratory burst of neutrophils in whole blood from term newborn infants as well as in adults. The reduced respiratory burst by fMLP might be a mechanism to reduce the detrimental effects of uncontrolled inflammation during neutrophil migration.

Neutrophil Receptor Response to Bacterial N-formyl Peptides is Similar in Term Newborn Infants and Adults in Contrast to IL-8.

We have previously observed that neutrophils from neonates exhibit different migratory responses to intermediate and end-target chemoattractants compared to adults. The aim of this study was to investigate the effect of the chemoattractants IL-8 (intermediate) and formyl-methionyl-leucyl-phenylalanine (fMLP; end-target) on cell surface receptor expression involved in adhesion, migration and granule release of neutrophils from term newborn infants and adults. Heparinized cord blood from 16 healthy term newborn infants delivered by caesarean section and peripheral blood from 17 healthy adults were incubated with 1 µm IL-8 or 0.1 µm fMLP, previously defined as optimal inducers of neutrophil migration. The leukocytes were labelled with antibodies to cell surface receptors (CD11b, CD15S, CD18, CD35, CD44, CD64, CD65, CD88, CD162, CD181 and CD182). Receptor expression was quantified by flow cytometry analysis. Upregulation of CD11b and downregulation of CD88 and CD182 after stimulation with IL-8 were more pronounced in adults than in neonates (P < 0.05, P < 0.05 and P ≤ 0.001, respectively), whereas fMLP induced changes in receptor expression that were of the same magnitude in neutrophils from neonates as from adults. We observed similar expression of receptors that mediate adhesion, migration, granule activation and phagocytosis induced by fMLP in neutrophils from neonates and adults. In contrast, differences between neonates and adults, induced by IL-8, suggest that the neutrophil response to intermediate chemoattractants might lead to a compromised infectious response in newborn infants.

Regulation of granulocyte function by hyaluronic acid. In vitro and in vivo effects on phagocytosis, locomotion, and metabolism.

Hyaluronic acid (HA) stimulated the function of polymorphonucler leukocytes (PMN) both in vitro and in vivo. Stimulation in vitro was achieved by the incubation of PMN and HA in heparinized whole blood at concentrations of HA between 5 and 500 microgram/liter. The stimulation of the PMN function was demonstrated by an increase rate of phagocytosis of complement- and/or immunoglobulin (Ig)G-coated latex particles, increased adherence to nylon wool, increased random migration and chemotactic response, increased chemiluminescence during phagocytosis, and raised levels of intracellular ATP. The effect of HA in vivo was demonstrated, after subcutaneous administration of HA (5-20 mg) to healthy volunteers, by an enhanced rate of phagocytosis of the subsequently isolated neutrophils. The duration of the effect of one administration was approximately 1 wk with maximum effect on days 2-4. HA injections to patients with increased susceptibility to bacterial infections and impaired neutrophil function demonstrated an enhanced neutrophil function also in these individuals. HA may therefore be a new principle by which resistance to infections can be enhanced.

The chemotactic response of granulocytes to the low molecular weight chemoattractants f-MLP, C5f, and LTB4 is dependent on chemokinetic factors.

The dependence of the low molecular weight chemoattractants (LMCs) formylmethionyl-leucylphenylalanine (f-MLP), C5f, and leukotriene B4 (LTB4) on albumin to express their chemotactic activity towards granulocytes (PMNs) was investigated in order to study the required qualities of albumin and if albumin could be replaced by any other proteins. The results demonstrated that the supporting effect of isolated albumin was dependent on the method of purification. Only isolated albumin exposed to ethanol precipitation during the purification procedure supported the chemotactic effect of LMCs. The albumin preparation that supported the effect of LMCs also mediated a chemokinetic effect on PMN migration. Albumin isolated by methods other than ethanol precipitation neither exerted a chemokinetic effect nor supported the chemotactic effect of LMCs. Heated, normal serum and isolated alpha 1-antitrypsin supported the chemotactic activity of LMCs and also mediated a chemokinetic effect on PMN migration. The present investigation suggests an important role for the chemokinetic factors, since it is indicated that their presence is necessary for the chemotactic response of PMNs to the low molecular weight chemoattractants C5f; LTB4, and f-MLP.

Inhibition of neutrophil and eosinophil chemotactic responses to PAF by the PAF-antagonists WEB-2086, L-652,731, and SRI-63441.

The influence of the three PAF-antagonists WEB-2086, L-652,731, and SRI-63441 on the chemotactic response of neutrophil and eosinophil granulocytes to PAF was investigated. When the PAF-antagonists were added to the cell suspension that was exposed to a gradient of PAF, WEB-2086 and SRI-63441 at the concentration of 10(-6) mol/litre inhibited (P less than .01) the neutrophil and eosinophil chemotactic response to 10(-8) and 10(-9) mol PAF per litre; at the concentration of 5 x 10(-6) mol/litre, WEB-2086 and SRI-63441 also inhibited (P less than .02) the response to 10(-7) mol PAF per litre. Under the same conditions L-652,731 at the concentration of 5 x 10(-6) mol/litre inhibited (P less than .01) the eosinophil chemotactic response to 10(-8) and 10(-9) mol PAF per litre. The inhibition of the chemotactic response to PAF by the three PAF-antagonists was specific, since the chemotactic response to C5f, f-MLP, and LTB4 was not affected by WEB-2086, L-652,731, or SRI-63441, neither was the chemokinetic migration induced by albumin.

New method for the measurement of eosinophil migration.

A new application of the Boyden chamber method for the measurement of eosinophil migration, without the need of eosinophil isolation, has been developed. A cell suspension containing a mixture of granulocytes, neutrophils to the greater part, was used. The eosinophils were identified by staining their granules with Chromotrope 2R. The method made it possible to study the migration of eosinophils from normal individuals without eosinophilia. Control experiments demonstrated that the chemotactic and chemokinetic response of eosinophils in the granulocyte mixture was in accordance with the response of isolated eosinophils from the same donor. Normal eosinophils demonstrated a significant chemotactic response to C5f, platelet-activating factor (PAF), leukotriene B4 (LTB4), and f-meth-leu-phe (f-MLP). Furthermore, PAF was demonstrated to be significantly more eosinophil chemotactic than neutrophil chemotactic.

Mechanism of action of cholinesterase inhibitors in Alzheimer's disease.

Cholinesterase inhibitors, such as physostigmine and tacrine, have lately gained interest as potential drugs in the treatment of Alzheimer's disease. Already in the 1950s, it was discovered that physostigmine and tacrine were potent inhibitors of acetylcholinesterase and butyrylcholinesterase. However, later studies have shown that cholinesterase inhibitors also interact with cholinergic receptors, with sodium and potassium ion channels and effect the uptake, synthesis and release of neurotransmitters. In summary, cholinesterase inhibitors are drugs with many modes of action, which may be of advantage in the treatment of a complex disorder such as Alzheimer's disease.

Effect of IFN-alpha on tumor-infiltrating mononuclear cells and regressive changes in metastatic malignant melanoma.

Interferon-alpha (INF-alpha) has a documented activity against metastatic melanoma. To what extent an antiproliferative effect or tumor cell modulation or immunomodulation contributes to this antitumor effect is still uncertain. The role of immune mechanisms in the control of malignant melanoma is suggested by several studies. Therefore, this investigation used monoclonal antibodies, anti-CD4, anti-CD8, and anti-CD11c, to study the occurrence and distribution of tumor-infiltrating mononuclear cells in 10 untreated and 26 IFN-alpha-treated patients with regional metastatic malignant melanoma. IFN-alpha was given for 1-3 weeks before resection of the metastases. The infiltration of mononuclear cells in the stroma and close to tumor cells was studied. The duration of IFN-alpha treatment was found to be of importance for the immunomodulatory effect. In patients treated for < or = 1 week, tumor-infiltrating mononuclear cells were still mainly localized in the stroma, similar to the situation in untreated patients. The differences in CD4+ cells close to the tumor cells, comparing untreated patients and patients with various durations of IFN-alpha treatment, were highly significant (p = 0.009). Thus, IFN-alpha treatment resulted in recruitment of CD4+ cells close to the tumor cells. IFN-alpha had only a weak effect on the recruitment of CD8+ and CD11c+ mononuclear cells close to the tumor cells. Regressive changes in metastases were also analyzed and correlated to duration of treatment. Some of the criteria used for histopathologic regression in primary melanoma (distorted histologic architecture, low tumor cell density, and fibrosis) were applied to analyze the effect of IFN-alpha in metastatic melanoma. The tumor cell density was found to be significantly reduced in metastases with marked tumor regression compared with metastases with no, or only minor, regressive changes (p < 0.005). A chi-square analysis for trend, comparing untreated patients and patients with various durations of IFN-alpha treatment, showed that regressive changes of the tumor increased significantly during IFN-alpha treatment (p = 0.02).

Expression of ICAM-1 during IFN-alpha-based treatment of metastatic malignant melanoma: relation to tumor-infiltrating mononuclear cells and regressive tumor changes.

Intercellular adhesion molecule-1 (ICAM-1) plays an important role in cell to cell interactions. In malignant melanoma, ICAM-1 expression correlates with malignant behavior. We used monoclonal antibodies, anti-ICAM-1, anti-CD4+, anti-CD8+, and anti-CD11c+ to study the effect of interferon-alpha (IFN-alpha) on the expression of ICAM-1 by melanoma cells in regional metastases and its correlation to the occurrence of CD4+, CD8+, and CD11c+ cells close to tumor cells and in the tumor stroma. We also estimated the expression of ICAM-1 and regressive changes in malignant melanoma metastases, correlating the duration of treatment to these effects of IFN-alpha. Twenty-three IFN-alpha-treated and 10 untreated patients with regional metastatic malignant melanoma were studied. The duration of IFN-alpha treatment influenced the expression of ICAM-1. In metastases from patients treated for 1 week only, 1 of 5 showed high expression of ICAM-1 compared with 6 of 11 of those treated for 3 weeks (p = 0.01, chi-square test for trend comparing untreated patients and patients with various durations of IFN-alpha treatment). In IFN-alpha-treated patients with low expression of ICAM-1, none of 7 metastases showed CD4+ cells infiltrating close to tumor cells, in contrast to 6 of 10 metastases expressing high amounts of ICAM-1 (p = 0.03). Similarly, the expression of ICAM-1 was found to correlate with the occurrence of CD8+ cells close to the tumor cells (p = 0.04). We also showed a correlation between ICAM-1 expression and histologic evidence of tumor regression (p = 0.02).

The EORTC and drug development. European Organisation for Research and Treatment of Cancer.

Early drug development at EORTC has always been subject to structural changes to adapt to the rapid changes that occur in oncological drug development. The expertise of early drug developers has always been cross-fertilised with disease-/tumour-oriented groups and also backwards to the laboratory research groups. This results in the establishment of a solid and dedicated network of medical oncologists with focused expertise in cancer drug development. The EORTC Data Center is fully equipped with all expertise to support clinical research activities and includes regulatory, safety, and quality assurance desks. The EORTC New Drug development Programme (NDDP) provides methodological expertise to early clinical trials and coordinates phase I and phase II studies addressing various approaches. Through NDDP, the early clinical groups and the disease-/tumour-oriented groups have created specific networks to address early drug development in specific tumour types. This results in very efficient networks which have the resources and the patients to address and conduct challenging clinical trials in a standardised fashion ensuring the highest standards in cancer treatment.

Immunisation of metastatic cancer patients with MAGE-3 protein combined with adjuvant SBAS-2: a clinical report.

Fifty-seven patients with MAGE-3-positive measurable metastatic cancer, most of them with melanoma, were vaccinated with escalating doses of a recombinant MAGE-3 protein combined with a fixed dose of the immunological adjuvant SBAS-2, which contained MPL and QS21. The immunisation schedule included 4 intramuscular (i.m.) injections at 3-week intervals. Patients whose tumour stabilised or regressed after 4 vaccinations received 2 additional vaccinations at 6-week intervals. The vaccine was generally well tolerated. Among the 33 melanoma patients who were evaluable for tumour response, we observed 2 partial responses, 2 mixed responses and 1 stabilisation. Time to progression in these 5 patients varied from 4 to 29 months. In addition, a partial response lasting 10 months was observed in 1 of the 3 metastatic bladder cancer patients included. None of the tumour responses described above involved visceral metastases. Immunological responses to the vaccine will be reported separately.

Simultaneous analysis of eosinophil and neutrophil adhesion to plasma and tissue fibronectin, fibrinogen, and albumin.

A simple and convenient assay for the simultaneous measurement of eosinophil and neutrophil adhesion is described. Incubations were performed in microtitre plates coated with different proteins. Adhesion of eosinophils and neutrophils was determined by the use of specific radioimmunoassays for eosinophil cationic protein (ECP) and myeloperoxidase (MPO). Using this assay, Mn2+ induced a significant increase of the adhesion of eosinophils to plasma fibronectin and fibrinogen in a time-dependent fashion, while a small increase of the adhesion of neutrophils to these two proteins was observed. In contrast, a time-dependent potent increment of the adhesion of both eosinophils and neutrophils to tissue fibronectin and albumin was found. Tissue fibronectin preferentially supported eosinophil adhesion compared with that of neutrophils in the presence of Mn2+. PMA (10(-9) mol/l) induced a significant increase in the adhesion of eosinophils and neutrophils of the same pattern to all four proteins. However, when granulocytes were stimulated by Mn2+ in combination with PMA, eosinophils and neutrophils showed different patterns of response to plasma fibronectin and fibrinogen, respectively, but the same pattern of response to tissue fibronectin. f-MLP stimulated an early increase of the adhesion of neutrophils to fibrinogen, while a weak stimulation of the adhesion of eosinophils to plasma fibronectin and fibrinogen and of neutrophils to plasma fibronectin was observed. Co-stimulation with f-MLP and Mn2+ did not induce any additive effects on granulocyte adhesion. In conclusion, the assay allows rapid quantification of eosinophil and neutrophil adhesion and can be used to directly compare the response of neutrophils and eosinophils. The assay is thus suitable for studies aimed at identifying agents with a selective effect on either of the cells.

Measurement of neutrophil and eosinophil adhesion to E-selectin, VCAM-1, and ICAM-1 by the use of transfected fibroblast cell lines.

A method which enables the specific measurement of neutrophil and eosinophil adhesion to the endothelial cell adherence receptors E-selectin, VCAM-1 and ICAM-1 has been developed. The method is based on continuous cultures of cell lines of transfected hamster kidney fibroblasts (BHK-21), that selectively express each of the endothelial cell adhesions molecules. Isolated granulocytes are added to the cultured adherent fibroblasts at a ratio of 20:1 and the cells are coincubated for 60 min at 37 degrees C. After removal of the nonadherent granulocytes the amount of adherent granulocytes could be measured by addition of detergent and a peroxidase substrate. Selective measurement of neutrophil and eosinophil adhesion was accomplished by addition of detergent to the adherent cells, collection of extracts followed by measurement of the concentration of an eosinophil (eosinophil cationic protein) and a neutrophil (myeloperoxidase) granule protein, respectively, in the extracts. At basal conditions neutrophils and eosinophils showed significant adhesion to E-selectin and eosinophils a low degree of adhesion to VCAM-1. Significant adhesion of neutrophils and eosinophils to ICAM-1 and of eosinophils to VCAM-1 was selectively induced by addition of manganese ions (Mn2+) at a concentration of 0.5 mmol/l. Neutrophils demonstrated a significantly higher adhesion to E-selectin than eosinophils, while eosinophil adhesion to ICAM-I was significantly higher than that of neutrophils. In conclusion, a method to compare the adhesive capacity of neutrophil and eosinophil granulocytes towards specific endothelial cell adhesion molecules has been developed.

A method to study apoptosis in eosinophils by flow cytometry.

The aim of this study was to develop a simple flow cytometric procedure to study eosinophil apoptosis. Eosinophils were isolated from the peripheral blood of healthy, non-allergic individuals and then cultured in basal culture medium. The cells were examined after 24, 48 and 72 h for forward- and side scatter (FS-SSC) pattern, staining with FDA, PI, and anti-CD95, and light microscopic appearance. After culture for >24 h, two populations with different FS-SSC-patterns appeared, referred to as A and B. Population A consisted of living, FDA-positive eosinophils. The eosinophils in population B showed a lower FS scatter than those in population A and a staining pattern with PI indicating the presence of hypodiploid DNA. Anti-CD95 demonstrated a significant staining of the eosinophils in population B, which increased after 2 days in culture. The cells were sorted using a FACS-Scan cell sorter and by Annexin V-coated magnetic beads to permit separate analyses of PI-staining pattern, DNA electrophoresis, and light microscopic examination of the cells in population B. The present study suggest that it is possible to discriminate between apoptotic and living eosinophils using the FS-SSC pattern and the PI-staining pattern obtained by flow cytometry.

Spherex (degradable starch microspheres) chemo-occlusion--enhancement of tumor drug concentration and therapeutic efficacy: an overview.

The efficacy of systemic chemotherapy in the treatment of primary and secondary liver cancer is poor. Intra-arterial delivery of fluoropyrimidines resulted in a significantly higher tumor response, but survival was prolonged by only a few months. Obviously, there is still a great need for improved therapeutic strategies. As the regional blood flow is of importance for the advantage of intra-arterial administration of cytotoxic drugs, degradable starch microspheres (DSMs) have been developed specifically to achieve temporary vascular occlusion during coadministration of cytotoxic drugs. Peak plasma concentrations, as well as the area under the concentration-time curve (AUC) of mitomycin C in plasma have been found to be significantly reduced following intra-arterial coadministration with DSMs. Similar results were also obtained with other drugs, such as nitrosoureas, doxorubicin, and 5-fluorouracil. The temporary vascular occlusion induced by DSMs enables a coadministered drug to be lodged in the target area for a prolonged period of time, resulting in a selectively increased uptake of labeled low molecular weight markers and several cytotoxic drugs into liver tumors compared with normal liver tissue. Vascular occlusion induced by DSMs has been demonstrated to redistribute the blood flow to hypovascular areas, which might be of particular importance for improving the efficacy of intra-arterial chemotherapy of hypovascular liver tumors. Passage through arteriovenous shunts was generally increased after DSM injection. However, this was without clinical significance as respiratory distress symptoms were found in only 1% of the sessions and were not considered to be serious in any of these patients. To take advantage of the pharmacokinetic modulation of coinjected drugs and, in addition, the redistribution of blood flow to hypovascular tumor areas, the goal is to achieve an almost complete vascular occlusion by injection of DSMs. Therefore, due to the wide variation between patients in the size and vascularity of liver tumors, the dose of DSMs has to be individualized. Degradable starch microspheres have been shown to enhance the antitumor efficacy of several cytotoxic drugs in animal experimental models and in noncomparative and randomized clinical studies.

Priming of eosinophil and neutrophil migratory responses by interleukin 3 and interleukin 5.

In the present study the influence of interleukin 3 and interleukin 5 on the migration of normal eosinophil and neutrophil granulocytes has been investigated. LTB4, PAF, f-MLP, C5a and ZAS were used as chemoattractants, and HSA and pooled normal human serum were used as chemokinetic agents. Recombinant human IL-5 (rh-IL5) at a concentration of 4 x 10(-12) mol/l was chemotactic for eosinophils, while recombinant mouse IL-5 (rm-IL5) attracted both eosinophils and neutrophils. IL-3 (rh-IL3) at a concentration around 10(-12) mol/l exerted a priming effect on eosinophil and neutrophil migration, i.e. chemotactic and chemokinetic responses to all agents tested. Human IL-5 at a concentration of 2 to 20 x 10(-12) mol/l primed the chemotactic and chemokinetic responses of eosinophils to all agents tested. The migration of neutrophils was also primed by rh-IL5, but at higher concentrations, i.e. around 10(-10) mol/l. IL-5 of mouse origin primed the migration of both eosinophils and neutrophils. In conclusion, IL-3 primed the migratory function of both eosinophils and neutrophils, while IL-5 was a more potent primer of eosinophil than of neutrophil migration.

Monocytes, but not macrophages, produce the eosinophil cationic protein.

The eosinophil cationic protein (ECP) is a cytotoxic protein with ribonuclease activity, produced and stored in bone marrow eosinophil myelocytes. Mature circulating eosinophils contain about 10 pg ECP per cell. The aim of this study was to investigate the possibility that monocytes produce and store ECP. By results from flow cytometry and specific protein measurement it is shown that human monocytes contain ECP (monocytes about 10 fg ECP per cell). RT-PCR analysis indicated the presence of mRNA coding for ECP in blood monocytes but not in alveolar macrophages. Furthermore, mRNA coding for ECP and low amounts of the protein were found in three myeloid cell lines representing different stages of monocytic differentiation. Differentiation of U-937 cells to macrophages induced lowered transcription of the ECP gene and reduced protein production. Immunohistochemical staining of lung tissue indicated that lung macrophages do not contain ECP. It is concluded that ECP is produced to a low extent by human monocytes and that the production is shut down during macrophage differentiation. This might indicate an alternative transcriptional regulation of the ECP gene in the monocytic lineage compared to the eosinophil lineage.

Gangliosides in serum immune complexes from tumor-bearing patients.

Immune complexes in the serum of tumor-bearing patients were adsorbed from whole blood or plasma on a protein A-Sepharose column. The adsorbed material was eluted, precipitated and analyzed for gangliosides. All precipitates obtained from eight patients at different treatment occasions contained gangliosides at concentrations varying from 0.1 to 12.2 nmol sialic acid/mg protein. The compositions of gangliosides were similar among the patients, regardless of the type of cancer, and quite different from that of normal serum. Most (75-85% of total sialic acid) belonged to the gangliotetraose series, of which 26-33% was GM1, 26-34% GD1a, 8-17% GD1b, and 5-13% GT1b. However, the dominant ganglioside in normal serum, GM3, was present in only trace amounts, which ruled out a nonspecific adsorption of serum ganglioside by protein A-Sepharose. Similar results were obtained for whole blood and plasma treatments, and these results suggest a specific interaction between gangliosides of the gangliotetraose series and serum immunoglobulins, either by the gangliosides acting as antigens and forming immune complexes or by their binding to already formed complexes.

Effects of IGF-1, truncated IGF-1 and the tripeptide Gly-Pro-Glu on acetylcholine release from parietal cortex of rat brain.

The effects of intact IGF-1, truncated IGF-1 and Gly-Pro-Glu (GPE), the aminoterminal tripeptide of IGF-1, on the potassium (35 mM K+) stimulated release of acetylcholine (ACh) from rat cortical slices were investigated. GPE significantly increased the release of ACh in the dose range of 10(-10)-10(-6) M, while IGF-1 significantly enhanced the release of ACh only at 4 x 10(-8) M. The truncated form of IGF-1, lacking the tripeptide GPE, did not effect the release of ACh in rat cortex. Binding experiments also showed that truncated IGF-1 was less available to the brain slices. The possible underlying mechanisms of action of GPE in the cholinergic synapse were investigated. GPE (10(-5) M) significantly (40%) displaced [3H]nicotine from its binding sites in rat cortex. In the concentration range of 10(-10)-10(-5) M, GPE did not interact with the choline uptake sites ([3H]hemicholinium binding) or the muscarinic ([3H]QNB) receptor binding sites in rat cortex. The mechanism of action behind GPEs enhancement of cholinergic transmission is therefore still unknown.

Tetrahydroaminoacridine induces opposite changes in muscarinic and nicotinic receptors in rat brain.

Rats were treated with 10 mg/kg tetrahydroaminoacridine (THA) twice daily for 14 days. THA (10 mg/kg) induced a significant decrease in the number of muscarinic receptors (both M1 and M2) in the cortex and striatum, whereas the number of nicotinic receptors in the cortex and hippocampus increased. Rats treated with physostigmine (0.9 mg/kg) showed a reduced number of muscarinic receptors, but no change in nicotinic receptors. The results indicate that treatment with cholinesterase inhibitors can induce opposite changes in brain muscarinic and nicotinic receptors in vivo.