Effects of high- vs moderate-intensity exercise on natural killer cell activity

The effect of 45 min of high- (80% VO2max) vs moderate- (50% VO2max) intensity treadmill exercise on natural killer cell cytotoxic activity (NKCA) was investigated in 10 well-conditioned (66.0 +/- 1.9 ml.kg-1.min-1), young males (22.1 +/- 1.3 yr). Blood samples were taken before and immediately after exercise, with three more samples taken during 3.5 h of recovery, and analyzed for proportion of NK cells (CD3-CD16+CD56+) and NKCA. Exercise at 80% vs 50% VO2max resulted in a greater immediate postexercise increase in proportion of NK cells, followed by a 1-h and 2-h decrease below preexercise levels for both intensity conditions. NKCA rose significantly above preexercise levels following high- but not moderate-intensity exercise. For both exercise intensity conditions, NKCA tended to drop below preexercise levels by 1 h postexercise, rising back to preexercise levels by 3.5 h postexercise. When NKCA was expressed on a per-NK cell basis, however, no change relative to preexercise levels occurred following moderate-intensity exercise, while a significant increase occurred after 2-h recovery from high-intensity exercise. These data demonstrate that both high- and moderate-intensity exercise are associated with significant shifts in circulating proportions of NK cells which significantly influence interpretation of NKCA data based on assays using separated mononuclear cells.     

Effect of a Staphylococcus epidermidis-extracted slime factor on human natural killer cell activity

A slime factor produced by Staphylococcus epidermidis was a complex glycoconjugate extracted by the phenol extraction method. The potential stimulatory or inhibitory capacity of the phenol-extracted slime (PES) was tested on human natural killer cell cytotoxic activity. Various concentrations of the PES preparation were incubated with the effector cells 30 min before and during the assay period. The PES factor inhibited natural killer cell cytotoxic activity at a concentration of 250 micrograms/ml and at higher concentrations (p < 0.05). The inhibition of natural killer cell cytotoxic activity may probably be related to the complex composition of the slime substance.     

In vitro and clinical effect of pentoxifylline on natural killer cell activity

Pentoxifylline used in the treatment of vascular diseases has also some immunological effects. Among of these effects the influence of pentoxifylline on the natural killer cell activity was studied. In in vitro experiments the human natural killer cell cytotoxicity was examined in the presence of pentoxifylline. In our clinical trial we investigated the topic whether this drug has an immunomodulatory effect while administering it for different periods. The natural cytotoxicity of macroangiopathic patients treated with pentoxifylline was compared with the values of healthy controls and patients suffering from vascular disease and obtaining no pentoxifylline therapy. Sixty two macroangiopathic patients and twenty healthy controls were investigated altogether. In the in vitro natural killer cell reaction we found that the pentoxifylline was able to suppress the cytotoxicity at any time of the reaction. The influence of pentoxifylline on natural killer cell activity was neither due to the expression of intercellular adhesion molecule-1, nor to the alteration of membrane fluidity. However, this drug significantly (p < 0.05) decreases the apoptosis of target cells. The natural killer cell activity of patients with chronic pentoxifylline therapy lasting for more than a year was proved to be significantly (p < 0.005) lower. The presence of vascular disease does not influence the natural killer activity by itself. Concluding our results we can state that the suppressing effect of pentoxifylline on natural killer cell activity needs to be taken into consideration in case of clinical diseases where this drug is administered chronically.     

Effect of clarithromycin treatment of natural killer cell activity in patients with advanced non-small cell lung cancer]

The treatment of a 14-membered ring macrolide, clarithromycin (CAM), prolongs the survival time of patients with unresectable nonsmall cell lung cancer, and improves the host factor. As we previously reported, one of the underlying mechanisms is that the treatment of CAM increases the bioactivity of interleukin-12 (IL-12). In the present study, we administered CAM to murine lung cancer treatment models with Lewis lung carcinoma and to 18 patients with unresectable non-small lung cancer whose anticancer treatment had been terminated. The timing of CAM administration was examined and the time course of NK activity was measured. In the murine lung cancer treatment models, administration of CAM 7 days after anticancer chemotherapy more strongly inhibited the tumor growth and more rapidly and significantly increased NK activity, compared to the concomitant use of CAM with an anticancer chemotherapy. In humans, the NK activity which had decreased after anticancer treatment, tended to be increased after one month of treatment with CAM (p = 0.06). One month of treatment with CAM significantly increased the NK activity (p < 0.05) of the following subjects: patients with stage III in the clinical stages, patients with squamous cell carcinoma, patients who had received radiotherapy alone as pretreatment therapy, and patients whose pretreatment therapy effect was partial response (PR). We conjectured that increasing NK activity was one of the underlying mechanisms of the macrobiotic effect of CAM. CAM was especially effective for patients in the early clinical stages and patients who responded well to pretreatment therapy. Murine lung cancer models showed that non-concomitant use of CAM with anticancer chemotherapy was more effective.     

Antileukemia activity of a natural killer cell line against human leukemias

We describe here the in vitro and in vivo antileukemia activity of a recently described natural killer (NK) cell line (NK-92), which has features of human activated NK cells. The cytotoxic activity of rhIL2-dependent cultured NK-92 cells against primary patient-derived leukemic target cells [12 acute myelogenous leukemias (AMLs), 7 T acute lymphoblastic leukemias (T-ALLs), 14 B-lineage-ALLs, and 13 chronic myelogenous leukemias (CMLs)], human leukemic cell lines (K562, KG1, HL60, Raji, NALM6, TALL-104, CEM-S, and CEM-T) and normal bone marrow cells was measured in 51Cr-release assay (CRA). The patient-derived leukemias could be subdivided into three groups based on their sensitivity to NK-92 cells: insensitive (< or =19% lysis), sensitive (20-49% lysis), and highly sensitive (> or =50% lysis) at an E:T ratio of 9:1. Of 46 patient-derived samples, 24 (52.2%) were sensitive or highly sensitive to NK-92-mediated in vitro cytotoxicity (6 of 12 AMLs, 7 of 7 T-ALLs, 5 of 14 B-lineage-ALLs, and 6 of 13 CMLs). NK-92 cells were highly cytotoxic against all of the eight leukemic cell lines tested in a standard 4-h CRA. Normal human bone marrow hematopoietic cells derived from 18 normal donors were insensitive to NK-92-mediated cytolysis. In comparison with human lymphokine-activated killer cells, normal NK cells, and T cells, NK-92 cells displayed more powerful antileukemia activity against a patient-derived T-ALL as well as K562 and HL60 cells, both in in vitro CRA and in a xenografted human leukemia SCID mouse model. The NK-92 cells did not induce the development of leukemia in SCID mice after i.v., i.p., or s.c. inoculation. In adoptive transfer experiments, SCID mice receiving i.p. inoculations of human leukemias derived from a T-ALL (TA27) and an AML (MA26) that were highly sensitive to the cytolysis of NK-92 cells in vitro, as well as a pre-B-ALL (BA31) that was insensitive to the in vitro cytolysis of NK-92 cells, were treated by administration of NK-92 cells with or without rhIL2 (2 x 10(7) NK-92 cells i.p.; one dose or five doses). Survival times of SCID mice bearing the sensitive TA27 and MA26 leukemias were significantly prolonged by adoptive cell therapy with NK-92 cells. Some of the animals who received five doses of NK-92 cells with or without rhIL2 administration were still alive without any signs of leukemia development 6 months after leukemia inoculation. In contrast, survival of mice bearing the insensitive BA31 leukemia were not affected by this treatment. This in vitro and in vivo antileukemia effect of NK-92 cells suggests that cytotoxic NK cells of this type may have potential as effectors of leukemia control.     

FK506 promotes liver regeneration by suppressing natural killer cell activity

We examined the mechanism of promotion of liver regeneration by tacrolimus hydrate (FK506), a potent immunosuppressant, after partial hepatectomy. The administration of FK506 significantly increased the bromodeoxyuridine (BrdU) labelling index at 36 and 48 h after 70% hepatectomy compared with the placebo group. Using the same model, we examined the effect of FK506 on the expression of hepatocyte growth factor (HGF) and transforming growth factor-beta 1 (TGF-beta 1) and found no changes in HGF and TGF-beta 1 mRNA expression in the liver or in the HGF protein concentration in plasma. We found that pretreatment with FK506 markedly reduced the activity and number of liver-resident natural killer (NK) cells at the time of partial hepatectomy. Our observations suggest that the promotion of liver regeneration by FK506 may be attributable to a reduction in the number of liver-resident NK cells and to inhibition of their activity.     

Expression of killer cell inhibitory receptors on human uterine natural killer cells

The establishment of the human placenta in early pregnancy is characterized by the presence of large numbers of natural killer (NK) cells within the maternal decidua in close proximity to the fetally-derived invading extravillous trophoblast which expresses at least two HLA class I molecules, HLA-G and HLA-C. These NK cells have an unusual phenotype, CD56(bright) CD16, distinguishing them from adult peripheral blood NK cells. They may control key events in trophoblast migration and therefore placentation. Human NK cells in peripheral blood express receptors for polymorphic HLA class I molecules. This family of receptors, known as killer cell inhibitory receptors (KIR), are expressed on overlapping subsets of NK cells to give an NK cell repertoire which differs between individuals. Using a panel of monoclonal antibodies to several members of the KIR family and analysis by flow cytometry, we have found that KIR are expressed by decidual NK cells. There is variation in both the percentage of cells expressing a particular receptor and the density of receptor expression between decidual NK cells from different individuals. Comparison of NK cells from decidua and peripheral blood of the same individual showed that NK cells from these two different locations express different repertoires of KIR. Receptors are present in individuals who do not possess the relevant class I ligand, raising the possibility that these NK receptors may be involved in recognition of the allogeneic fetus by the mother at the implantation site.     

Chronic stress and natural killer cell activity after exposure to traumatic death

The beta recombinase encoded by the streptococcal plasmid pSM19035, which shows 28 to 34% identity with DNA resolvases and DNA invertases, can catalyze formation of deletions or inversions between properly oriented target sites. We have constructed a number of site-directed mutations at residues that are conserved between the beta protein and other DNA recombinases of the resolvase/invertase family. The analysis of the recombination and DNA-binding ability of each mutant protein shows that the mutations affect the catalytic activity and, in two cases, the dimerization of the protein. The results suggest that the beta protein probably mediates recombination by a catalytic mechanism similar to that proposed for the resolvase/invertase family. Since the beta recombinase differs from DNA resolvases and DNA invertases in its lack of bias towards either of these reactions, the results presented support the hypothesis that its unique properties might depend on details of the architecture or assembly of the recombination complex. In addition, two beta protein mutants that can no longer form dimers in solution have provided new insights into the way the protein binds to DNA.     

Acute alcohol intoxication suppresses natural killer cell activity and promotes tumor metastasis

Alcohol consumption is associated with increased morbidity and mortality related to infectious diseases and malignancy (1-5), although immune mediation of these relationships is controversial. Specifically, the activity of natural killer (NK) cells, which are involved in the resistance to infections and metastasis, can be suppressed in the presence of ethanol in vitro. However, acute consumption or infusion of ethanol in vivo exerts no effects on NK activity assessed in vitro thereafter. Therefore, we have developed and used a method to study the effects of ethanol on NK activity in living rats by using an NK-sensitive metastatic process and selective depletion of NK cells in vivo. Acute ethanol intoxication caused a marked suppression of NK activity in vivo and a tenfold increase in the number of MADB106 tumor metastases. Ethanol had no effect in rats selectively depleted of NK cells or when an NK-insensitive tumor (C4047) was used. These findings suggest that even acute ethanol intoxication markedly suppresses NK activity in the living organism. This suppression may underlie some aspects of the association between alcoholism, infectious disease and malignancies.     

IFN-gamma-inducing factor up-regulates Fas ligand-mediated cytotoxic activity of murine natural killer cell clones

NK cells, non-T non-B immune effector lymphocytes, are localized in many organs, including liver, as well as in the circulation. To investigate the regulatory mechanism of killing apparatus in hepatic NK cells, we established IL-2-dependent NK cell clones from liver lymphocytes of BALB/c nude mice. To generate the NK cell clones, we incubated liver lymphocytes with a high dose of IL-2 in the presence of irradiated Kupffer cells, as feeder cells and as the source of IL-12, originally identified as NK cell stimulatory factor. Unless liver lymphocytes were incubated with both IL-2 and Kupffer cells, no cell growth was observed. Hepatic NK cell clones were established from this cell line by limiting dilution. The surface phenotypes of cloned NK cells were IL-2R beta-chain+ CD16+ CD3- IgM-. The clones did not express NK2.1, which is expressed by a half of NK-enriched spleen cells of BALB/c mice. Although the cells contained dense granules reactive to mAb against perforin, they exerted no conventional cytolytic activity against YAC-1. They constitutively expressed Fas ligand (FasL) and specifically killed Fas-positive target cells by fragmenting DNA. This Fas-FasL-mediated killing activity was enhanced by IFN-gamma-inducing factor, a recently identified novel cytokine produced by activated Kupffer cells, but was not affected by other Kupffer cell-produced cytokines, such as IL-12, IL-1beta, and TNF-alpha. Taken together, these findings suggest that hepatic NK cells participate in the immune response as effector cells through the Fas-FasL system in collaboration with cytokines from Kupffer cells.     

Self-discrepancy and natural killer cell activity: Immunological consequences of negative self-evaluation.

Tested whether self-discrepancy theory could account for changes in natural killer (NK) cell activity after exposure to self-referential stimuli. Anxious, dysphoric, and control Ss were pretested and 1 mo later covertly exposed to their own self-guides as well as those of another S. Blood samples were drawn for analysis of NK cytotoxicity and cortisol. The dysphoric Ss manifested the greatest actual:ideal discrepancy, whereas the anxious Ss manifested the greatest actual:ought discrepancy. Content analysis of written responses showed that activating discrepancies induced specific negative states; priming discrepancies also increased cortisol for the anxious Ss. NK activity was lower after self-referential priming for both distressed groups, particularly the anxious Ss. The control Ss showed a trend toward increased NK activity after self-referential priming. The study represents the 1st experimental demonstration that negative self-evaluation can alter immune responses. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Expression of different lipoprotein receptors in natural killer cells and their effect on natural killer proliferative and cytotoxic activity

Natural killer (NK) cells take up chylomicrons (CM), very low density (VLDL), low density (LDL), high density (HDL) and acetyl-modified low density (AcLDL) lipoproteins through different receptors, VLDL being the lipoprotein with the highest uptake and HDL the lowest. The uptake of LDL can be selectively blocked by the anti-LDL receptor, which does not affect the uptake of CM, VLDL, HDL and AcLDL. Although the uptake of lipoproteins assessed by flow cytometry using DiI is not very high, the lipoproteins are able to induce an increase in proliferative responses, VLDL, AcLDL and HDL being the most important ones with 12- and 17-fold increments, respectively. CM, VLDL and LDL at low concentrations increase NK cytotoxic activity, while HDL and AcLDL inhibit, in a dose-dependent fashion, the killing of NK cells against K562. These results suggest the presence of four different receptors that are responsible for the cytotoxic and proliferative responses observed.     

Inhibition of natural killer cell activity in mice treated with tobacco specific carcinogen NNK

Among the different chemicals present in tobacco and tobacco smoke, 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen. In the present study the immunosuppressive effect of NNK was investigated in laboratory animals by analyzing the antitumor immune responses. Mice of B6C3F1 strain were treated with different doses of NNK by IP and assayed for natural killer cell activity by the lysis of 51Cr-labeled YAC-1 lymphoma cells. The control mice received physiological saline. The results showed a significant inhibition of natural killer cell activity in the spleen cells of mice treated with 100 or 250 mg/kg NNK. In contrast to the high-dose NNK group, treatment of mice with lower doses of NNK like 10 or 50 mg/kg had no significant effect on the natural killer cell activity. In addition to spleen, the natural killer cell activity was also suppressed in the hilar lymph nodes and lung cells of NNK-treated mice. The clearance of 125I labeled YAC-1 tumor cells was also reduced from the lungs of mice injected with NNK. Further, the metastatic potential of B16F10 melanoma cells was significantly higher, as evidenced by the increased lung tumor nodules in the high-dose NNK-treated mice. The decreased antitumor immune response in the carcinogen-treated mice was not due to a decrease of NK cells, because flow cytometric analysis indicated no change in the frequency of NK 1.1+ cells between control and treated animals. However, there was an increased plasma cortisone levels in the carcinogen-treated mice compared to control animals. Injection of mice with poly I:C or interleukin-12 was able to restore natural killer cell activity in the tobacco carcinogen-treated mice.     

Inhibition of natural killer cell cytotoxicity by cell growth-related molecules

Certain MHC class I molecules on target cells are known to inhibit the cytotoxic action of NK cells. By using monoclonal antibody (mAb) Cho-1, we have found inhibitory non-MHC class I cell surface molecules that are noncovalently-associated with 200 kDa and 40 kDa antigens. Poly I-C-induced rat NK cells were not cytotoxic to rat fetus-derived fibroblast WFB cell line. In contrast, NK cells were cytotoxic to H-ras oncogene-induced transformants of WFB, W14 and W31. FACS analysis indicated that mAb Cho-1 reacts with WFB, but not with W14 and W31 cells. Thus, this antigen may disappear concomitantly with cell growth and transformation. Cho-1 antigens were also expressed on other NK-resistant lines, such as mouse BALB3T3 fibroblast, EL-4 lymphoma and human fibroblast HEPM. However, they were not expressed on NK-sensitive mouse YAC-1 and H-ras transformant (Brash) of BALB3T3 cells. Furthermore, treatment of target cells with IFN-gamma clearly induced the cell surface expression of Cho-1 antigens, and conferred a resistance to NK cytolysis on target cells. These data strongly suggest that Cho-1 antigen expression may correlate with target cell susceptibility to NK cells. Indeed, treatment of NK-resistant WFB as well as HEPM cells with F(ab')2 fragments of mAb Cho-1 resulted in the acquisition of susceptibility to NK cytolysis. Cho-1 antigens may be novel molecules that regulate the NK resistance of cells.     

Changes with aging, sex and physical exercise in murine natural killer activity and antibody-dependent cellular cytotoxicity

Antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) activity were measured in leukocytes from the axillary nodes, the spleen and the thymus of young (12 +/- 2 weeks) and aged (60 +/- 2 weeks) male and female BALB/c mice, which had performed an acute bout of exercise (moderate swimming until exhaustion) or a training exercise (90 min of moderate swimming each day for 20 days). The results show that NK and ADCC activity in sedentary mice (controls) were similar in young and aged animals. However, both kinds of exercise resulted in higher cytotoxicity values in aged mice than in young mice. Acute exercise did not have any effect on NK activity in young and aged mice, nor on ADCC activity in young mice as compared to controls, while training exercise stimulated both cytotoxicities in the two age groups. No correlations between serum corticosterone levels and NK or ADCC activity were found. Our results suggest that moderate training exercise improves both NK and ADCC activity during aging.     

Augmentation of natural killer cell activity in mice by oral administration of transforming growth factor-beta

OBJECTIVE: To analyze the effect of HLA-DR genes on susceptibility to and severity of ankylosing spondylitis (AS). METHODS: Three hundred sixty-three white British AS patients were studied; 149 were carefully assessed for a range of clinical manifestations, and disease severity was assessed using a structured questionnaire. Limited HLA class I typing and complete HLA-DR typing were performed using DNA-based methods. HLA data from 13,634 healthy white British bone marrow donors were used for comparison. RESULTS: A significant association between DR1 and AS was found, independent of HLA-B27 (overall odds ratio [OR] 1.4, 95% confidence interval [95% CI] 1.1-1.8, P = 0.02; relative risk [RR] 2.7, 95% CI 1.5-4.8, P = 6 x 10(-4) among homozygotes; RR 2.1, 95% CI 1.5-2.8, P = 5 x 10(-6) among heterozygotes). A large but weakly significant association between DR8 and AS was noted, particularly among DR8 homozygotes (RR 6.8, 95% CI 1.6-29.2, P = 0.01 among homozygotes; RR 1.6, 95% CI 1.0-2.7, P = 0.07 among heterozygotes). A negative association with DR12 (OR 0.22, 95% CI 0.09-0.5, P = 0.001) was noted. HLA-DR7 was associated with younger age at onset of disease (mean age at onset 18 years for DR7-positive patients and 23 years for DR7-negative patients; Z score 3.21, P = 0.001). No other HLA class I or class II associations with disease severity or with different clinical manifestations of AS were found. CONCLUSION: The results of this study suggest that HLA-DR genes may have a weak effect on susceptibility to AS independent of HLA-B27, but do not support suggestions that they affect disease severity or different clinical manifestations.     

Suppression of natural killer cell activity in mouse spleen lymphocytes by several dopamine receptor antagonists

Glomerular diseases are associated with changes in glomerular membrane permeability properties that alter filtration rate of plasma water and barrier function of the capillary wall. To estimate intrinsic permeability properties that regulate transmembrane transport of water and macromolecules, theoretical analysis of renal clearance of tracer molecules can be used. The development of adequate theoretical models is required to achieve sufficient accuracy to simulate complicated biological processes. Current research in this area is aimed at improving and validating the presently available models in order to characterize the nature of permeability changes associated with pathological conditions. This is a key step in understanding the pathophysiological nature of glomerular diseases and in the development of effective treatments.     

Immunity and depression: insomnia, retardation, and reduction of natural killer cell activity

The contribution of chloride to the haemodynamic changes of salt-dependent deoxycorticosterone (DOC) hypertension was studied in young Wistar rats subjected to dietary loading with sodium chloride (NaCl) or sodium bicarbonate (NaHCO3). Mean arterial pressure (MAP), cardiac output, systemic resistance (TPR) and arterial rigidity (estimated from pulse pressure/stroke volume ratio, PP/SV) were determined in conscious chronically cannulated rats. DOC-induced increase of MAP and TPR appeared earlier in NaCl-loaded than in NaHCO3-loaded rats. After 4-6 weeks of hypertensive treatment MAP, TPR and PP/SV ratio were higher in DOC-treated rats fed NaCl diet than in those fed NaHCO3 diet. In contrast, after a long-term hypertensive regimen (lasting for 7-9 weeks) there was no significant difference in either MAP or TPR between rats loaded with NaCl or NaHCO3. On the other hand, DOC hypertension induced by a long-term feeding of NaHCO3 diet was not associated with an increase of arterial rigidity which was characteristic for DOC-NaCl hypertensive rats. Thus, a sufficiently long selective dietary sodium loading is capable to increase the systemic resistance but not to alter the arterial rigidity. This was also confirmed by a comparison of blood pressure-matched DOC hypertensive rats fed NaCl or NaHCO3 diets. These animals did not differ in the degree of systemic resistance elevation but the arterial rigidity was increased only in NaCl-loaded rats.     

The effects of general anesthesia and surgery on basal and interferon stimulated natural killer cell activity of humans

Natural killer (NK) cells can lyse certain tumor cells as well as virally infected cells without prior sensitization. Animal studies have shown that general anesthesia (GA) alone or GA followed by surgery decreases both basal NK cytotoxicity and enhancement of NK activity by interferon (IFN). The purpose of this study was to determine whether similar inhibition of NK activity by anesthesia and surgery occurs in humans. Venous blood was drawn 1 h before and 20-24 h after surgery under isoflurane/N2O anesthesia. Peripheral blood mononuclear cells (PBMC) were assayed for basal and IFN-alpha-stimulated NK cytotoxicity using a chromium release assay with K562 cells as targets. Flow cytometry was used to enumerate NK, T-helper, and T-cytotoxic/suppressor cell populations in each sample. Basal NK activity was significantly depressed after GA and GA and surgery. Although the postoperative IFN treatment increased NK activity to the preoperative basal level, the level achieved was significantly lower than the level observed after IFN stimulation of PBMC as evaluated preoperatively. This decreased activity does not seem to be the result of a decrease in the percentage of circulating NK cells. The decrease in NK activity after anesthesia and surgery may lead to increased susceptibility to infection and/or tumor dissemination and thus needs to be explored. Implications: Natural killer cells can kill cancer cells and virally infected cells. This study shows that surgery with general anesthesia leads to decreased natural killer cell activity as assessed int he laboratory. This decreased natural killer cell activity may lead to infection or tumor dissemination. NK activity can be restored to presurgery levels by treating isolated NK cells with interferon-alpha.     

Preoperative natural killer cell activity: correlation with distant metastases in curatively research colorectal carcinomas

A new technique for collecting blood from conscious and unrestrained rats is reported. Plasma arginine vasopressin (AVP) concentration has been known to be influenced by various procedures used for blood collection (e.g., manual contact and anesthesia). In the study reported here, the improvement of the routine jugular vein cannula method was undertaken by extending the connection tubing to allow use of a swivel mechanism. Plasma AVP, glucose, and corticosterone concentrations in virgin rats were measured in blood samples collected by use of the new method and of two generally accepted methods (decapitation and routine jugular vein cannula methods). All values of the three parameters obtained were the lowest when the new method was used, suggesting that this new swivel method induced the least stress in the rats.