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D L Evans

Publications and source records attributed to D L Evans.

At least 37 records · Page 2Linked to original sources

Detection and diagnosis of psychiatric disorders in primary medical care settings.

Research efforts accelerated in the 1990s to define the presentation of common psychiatric disorders in primary care settings. Two diagnostic instruments, the DSM-IV-PC and the PRIME-MD, were introduced in 1994, and a self-report form of the PRIME-MD, the PHQ, was published in 1999. These tools have streamlined the larger, often cumbersome psychiatric nomenclature of the DSM-III and DSM-IV and appear to be more useful in general medical settings. It still is not practical to use either instrument in its entirety for all patients in a busy primary care practice. Studies have suggested an efficient and effective, two-step method of screening primary care patients for psychiatric disorders, however. In this approach, a limited number of probing questions extracted from the PRIME-MD or PHQ (or DSM-IV-PC) are posed to patients, either in person or by a written self-report (i.e., a general health update or review of systems). Then a follow-up evaluation is done to confirm or refute positive screening results. Short, simple questionnaires that address specific topics (e.g., CAGE for alcohol screening or the GDS for mood disorders in older adults) complete and complement this approach. This method has the advantage of being easy to incorporate into routine office practice using minimal physician or office staff time, while showing acceptable sensitivity and specificity in studies to date. More research, particularly prospective studies, is needed to confirm the effectiveness of this approach and expand it beyond the few available studies that have focused mostly on depressive disorders.

Adult↗

A streamlined method for diagnosing common psychiatric disorders in primary care.

Primary care practitioners treat the majority of patients with psychiatric disorders, but many patients in general medical practices continue to suffer from undiagnosed mental illnesses. Over the past 10 years, clinical investigators have focused on enhancing the detection of psychiatric disorders in primary care. Several diagnostic tools have been developed for use by clinicians who are not psychiatrists. However, the time constraints of real-world office practice and the often-cumbersome psychiatric nomenclature have limited their use. Recent studies have tested even more streamlined diagnostic methods that require only a few minutes of physician time while correlating strongly with formal psychiatric evaluations. This article consolidates these research findings with practical experience in the rapid detection of 5 psychiatric conditions commonly encountered in primary care: depression, anxiety, stress reactions, substance abuse, and cognitive impairment. An efficient, 2-step method is described that consists of carefully targeted screening questions followed by a confirmatory evaluation. The screening questions and confirmatory examination can be incorporated easily into most primary care practices.

Aged↗

Doppler sonographic measurements of arterial blood flow and their repeatability in the equine foot during weight bearing and non-weight bearing.

In six normal adult horses, pulsed-wave Doppler sonographic inspections were made of the left and right lateral palmar proper digital artery (LPPDA) of each thoracic limb. Using a two-period, crossover design with repeated measures, each LPPDA was inspected on four occasions throughout the day over a 7 hour period and, on each occasion, with the limb weight bearing and non-weight bearing. In comparison with the observations made during weight bearing, there were significant increases (P<0.001) in blood-flow velocities when the limb was non-weight bearing. There was no relationship between blood velocities and the time of day, the order in which the inspections were made, resting heart rate, age of the horse, or hoof angle. In both weight bearing and nonweight bearing, the Doppler waveform demonstrated a systolic peak followed by at least two diastolic peaks and an end diastolic plateau. Horses that moved least during sonographic inspections had the lowest co-efficients variation.

Animals↗

Psychosocial-immune relationships in HIV disease.

Human immunodeficiency virus (HIV) is now commonly viewed as a chronic disease, which often consists of a wide array of recurrent and sometimes severe psychosocial stressors. An individual's response to these multiple challenges over time may impact their health. In this article, we review research examining the relationship of psychologic factors (eg, depression, stressful life events, coping, social support) with immune system function and disease course. We also explore some of the potential physiologic pathways that may underlie these types of psychosocial-immune relationships, as well as the effects of psychologic interventions, particularly cognitive-behavioral stress management (CBSM), on the psychosocial, neuroendocrine, and immune functioning of people living with HIV. We conclude by suggesting some areas for future research, particularly the study of HIV-positive women.

Adaptation, Psychological↗

Neuroimmunologic aspects of sleep and sleep loss.

The complex and intimate interactions between the sleep and immune systems have been the focus of study for several years. Immune factors, particularly the interleukins, regulate sleep and in turn are altered by sleep and sleep deprivation. The sleep-wake cycle likewise regulates normal functioning of the immune system. Although a large number of studies have focused on the relationship between the immune system and sleep, relatively few studies have examined the effects of sleep deprivation on immune parameters. Studies of sleep deprivation's effects are important for several reasons. First, in the 21st century, various societal pressures require humans to work longer and sleep less. Sleep deprivation is becoming an occupational hazard in many industries. Second, to garner a greater understanding of the regulatory effects of sleep on the immune system, one must understand the consequences of sleep deprivation on the immune system. Significant detrimental effects on immune functioning can be seen after a few days of total sleep deprivation or even several days of partial sleep deprivation. Interestingly, not all of the changes in immune physiology that occur as a result of sleep deprivation appear to be negative. Numerous medical disorders involving the immune system are associated with changes in the sleep-wake physiology--either being caused by sleep dysfunction or being exacerbated by sleep disruption. These disorders include infectious diseases, fibromyalgia, cancers, and major depressive disorder. In this article, we will describe the relationships between sleep physiology and the immune system, in states of health and disease. Interspersed will be proposals for future research that may illuminate the clinical relevance of the relationships between sleeping, sleep loss and immune function in humans.

Major Depressive Disorder↗

Locomotion evaluation for racing in thoroughbreds.

The potential racing and locomotory profile of a Thoroughbred yearling should be taken into account for its training programme and racing career. A gait test has been designed to assist the trainer in this task. The aim of this study was to investigate the temporal and kinetic locomotory variables of Thoroughbreds at the gallop, in relationship to their racing ability. Thirty Thoroughbred horses in race training were tested at maximal speed during a training session. The training exercise consisted of a warming-up session at trot and canter for 10 min followed by a gallop session at increasing speed on a dirt track 1942 m long. The maximal speed was measured for the last 800 m before the finishing post. An acclerometric device attached to the girth provided quantitative information about the kinetic and temporal variables of the gallop such as: stride length (SL), stride frequency (SF), times elapsed between each hoof midstance phase (HIND, DIAGO, FORE), regularity of the strides (REG), mean vector of propulsion (VPROP), energy of propulsion (EPROP) and energy of loading (ELOAD). The performance records (number of wins, placings and average earning/start [PERF]) were used to analyse the relationship with the gait measurements. The mean maximum speed was 15.26 m/s. Several locomotory variables were significantly (P < 0.05) correlated to the gallop speed: SL (0.90), SF (0.75), DIAGO (0.42), REG (-0.47), VPROP (0.52), ELOAD (0.56) and EPROP (0.65). There were significant correlations between PERF and the following gait variables: REG (0.79), DIAGO (0.43), SF (0.42), SL (-0.32) and ELOAD (-0.40). The horses that won short distance races (< 1400 m) had a larger relative ground contact duration and higher stride frequency than horses that won in longer distance races. The gait test was easy to perform and provided useful locomotory variables that may be used to evaluate the racing ability of the Thoroughbreds in training.

Acceleration↗

Double-blind, placebo-controlled comparison of imipramine and paroxetine in the treatment of bipolar depression.

OBJECTIVE: This study compared the efficacy and safety of paroxetine and imipramine with that of placebo in the treatment of bipolar depression in adult outpatients stabilized on a regimen of lithium. METHOD: In a double-blind, placebo-controlled study, 117 outpatients with DSM-III-R bipolar disorder, depressive phase, were randomly assigned to treatment with paroxetine (N=35), imipramine (N=39), or placebo (N=43) for 10 weeks. In addition to lithium monotherapy, patients may have received either carbamazepine or valproate in combination with lithium for control of manic symptoms. Patients were stratified on the basis of trough serum lithium levels determined at the screening visit (high: >0.8 meq/liter; low: </=0.8 meq/liter). Primary efficacy was assessed by change from baseline in scores on the Hamilton Rating Scale for Depression and the Clinical Global Impression illness severity scale. RESULTS: Differences in overall efficacy among the three groups were not statistically significant. For patients with high serum lithium levels, antidepressant response at endpoint also did not significantly differ from placebo. However, both paroxetine and imipramine were superior to placebo for patients with low serum lithium levels. Compared to imipramine, paroxetine resulted in a lower incidence of adverse events, most notably emergence of manic symptoms. CONCLUSIONS: Antidepressants may not be useful adjunctive therapy for bipolar depressed patients with high serum lithium levels. However, antidepressant therapy may be beneficial for patients who cannot tolerate high serum lithium levels or who have symptoms that are refractory to the antidepressant effects of lithium.

Adult↗

Postprandial arterial vasodilation in the equine distal thoracic limb.

The effects of feeding on blood flow to the equine foot are poorly understood. In a temperature-controlled room, duplex Doppler ultrasonographic observations were made pre- and postprandially of the lateral proper palmar digital artery of 5 horses, randomly assigned to twice and 4 times daily feeding in an unbalanced 2 period crossover design. Arterial diameter and blood velocity were measured over 4 h and additional observations made of heart rate, blood pressure, total plasma protein, packed cell volume, plasma glucose and insulin. There was no effect of the feeding regimen on any variable. Postprandially, there were significant increases in arterial diameter, blood velocity, total plasma protein, plasma glucose and insulin; the other variables were unchanged. It appeared that the normal postprandial response was an increase in blood flow to the foot. The value of ultrasonography for noninvasive investigation of the peripheral vasculature of the conscious horse was established; and in the future it may have a role in the diagnosis of vascular diseases of the foot.

Animal Feed↗

Activation-induced programmed cell death of nonspecific cytotoxic cells and inhibition by apoptosis regulatory factors.

Nonspecific cytotoxic cells (NCC) are the teleost equivalent of mammalian lymphokine-activated natural killer cells. The cytotoxic activities of NCC are enhanced by stress-activated serum factors (SASF) present in tilapia acute-phase serum. In the present study purified NCC and xenogeneic target HL-60 tumor cells and nuclei were distinguishable in mixtures determined by flow cytometry. NCC activated by target HL-60 cells undergo activation-induced programmed cell death (AIPCD) during 12- to 16-h killing assays as shown by Annexin-V binding and nuclear DNA fragmentation results. Annexin-V binding studies also demonstrated that NCC kill HL-60 cells by an apoptotic mechanism. NCC are protected from AIPCD by 4-h preincubation in 50% SASF. Pretreatment also produced more than a fourfold increase in NCC cytotoxicity (effector/target (E:T) ratio = 100:1). In the absence of SASF preincubation, the percentage of apoptotic NCC increased from 8 to 91% at E:T ratios of 1:0 and 1:1, respectively. Kinetic studies (E:T = 10:1) demonstrated that the percentage of NCC exhibiting HL-60-dependent AIPCD increased between 0.1 and 12 h and then decreased inversely with total cell necrosis over the next 60 h. Preincubation of NCC with SASF protected NCC from AIPCD for over 72 h. Crosslinkage of the NCCRP-1 receptor with monoclonal antibody (mab) 5C6 produced AIPCD between 1 and 100 microg/mL mab concentrations. Preincubation with SASF completely protected NCC from mab 5C6-dependent AIPCD. SASF-mediated protection of NCC from AIPCD was dependent upon divalent cations, as demonstrated by increases in DNA hypoploidy of 38, 67, and 88% following preincubation in the presence of 10, 100, and 1000 microM EDTA, respectively. SASF also protected NCC from glucocorticoid- (i. e., dexamethasone) induced apoptosis. Combined, these results demonstrated that NCC activity is down-regulated by AIPCD. Release of SASF into the peripheral circulation may prevent negative regulation of NCC by AIPCD by increasing recycling capacity. Results are discussed in the context of the effects of acute stressors on innate immunity.

Acute-Phase Proteins↗

Comparison of pramipexole, fluoxetine, and placebo in patients with major depression.

Pramipexole, a dopamine D2 receptor agonist, was tested in 174 patients with major depression, with or without melancholia and without psychotic features. Three daily dose levels (0.375 mg, 1.0 mg, and 5.0 mg) were compared to fluoxetine (Prozac) at 20 mg and placebo in a randomized, double-blind, parallel-group study. After a 1 week placebo run-in period, patients were treated for 8 weeks, had a post-study follow-up (week 9), and were evaluated primarily with the Hamilton Psychiatric Rating Scale for Depression (HAM-D), the Montgomery-Asberg Depression Rating Scale (MADRS), and the Clinician's Global Impressions-Severity of Illness scale (CGI-SI). All patients who received one dose of study medication were included in the observed-case analysis (no missing data were replaced). Results indicated that by endpoint (week 8), patients receiving pramipexole at the 1.0 mg per day dose had significant improvement over baseline compared to the placebo group by measure of the HAM-D, MADRS, and CGI-SI. Significant improvement in this dose group was seen at other timepoints as well. The most obvious improvement was seen in the pramipexole 5.0 mg group, although a substantial dropout rate for this group precluded statistical tests vs. placebo late in the study. Patients taking fluoxetine also showed significant improvements at endpoint on the MADRS and earlier in the study on the HAM-D. No new or unusual safety concerns were generated during this study. Pramipexole helped safely alleviate the symptoms of depression at 1.0 mg per day and especially in those patients who could tolerate the escalation to 5 mg per day.

Adult↗

Efficacy of venlafaxine in geriatric depression.

Geriatric patients with major depression present clinical challenges not encountered in younger individuals, including a greater incidence of medical comorbidity, higher rates of multiple medication use, changes in drug metabolism due to age or physical illness, and increased sensitivity to antidepressant side effects. Nevertheless, successful treatment of depressive disorders in the elderly improves mental and physical functioning, decreases morbidity and perhaps mortality, and enhances quality of life. Recent research indicates that newer antidepressants are effective for late life depression and safer for older individuals. Among newer antidepressants, venlafaxine has a pharmacological profile that makes it an attractive choice for geriatric patients. It has limited potential to interact with other medications because it only weakly inhibits the cytochrome P450 system and binds to plasma proteins at a low level. Dosing may have to be adjusted for patients with renal failure, but typically not for those with liver disease or other medical conditions. Data from three double-blind and four open clinical trials support the safety and efficacy of venlafaxine for geriatric depression. Patients may experience transient, generally tolerable side effects such as insomnia, nausea, agitation, or dry mouth early in treatment, but more serious problems such as falls or cardiac rhythm disturbances seem to be rare. Treatment emergent hypertension occurs in a small percentage of older patients, generally at doses above 150 mg/day. Finally, emerging data suggest that venlafaxine may be effective for conditions such as stroke, anxiety, and neuropathic pain that frequently accompany depressive disorders in the elderly.

Aged↗

Physiological effects of electroconvulsive therapy and transcranial magnetic stimulation in major depression.

Major depressive episodes are associated with dysregulation of various physiologic systems. Antidepressant medications alter regulation of the hormonal and sleep systems. A thorough understanding of these changes may elucidate the pathophysiologic basis of the disorder [Amsterdam et al., 1989: Psychoneuroendocrinology 14:43-62], and interventions targeted directly at these systems are being increasingly recognized as possible treatments for depression [Wong et al., 2000: Proc Natl Acad Sci USA 97:325-330; Szuba et al., 1996: Proc Am Coll Neuropsychopharmacol Ann Meet]. These physiologic systems are regulated by the major neurotransmitters implicated in the etiology of mood disorders--norepinephrine, serotonin, and dopamine. Many of the hormones of import for this article also act as neurotransmitters and thus alter cerebral activity themselves [Owens and Nemeroff, 1993: Ciba Found Symp 172:296-308; Weitzner, 1998: Psychother Psychosom 67:125-132]. Parenteral infusion of hydrocortisone [DeBattista, 2000: Am J Psychiatry 157:1334-1337] and thyrotropin-releasing hormone (TRH) [Prange et al., 1972: Lancet 2:999-1002; Marangell et al., 1997: Arch Gen Psychiatry 54:214-222; Szuba, 1996: Proc Am Coll Neuropsychopharmacol Ann Meet.] produce acute antidepressant effects. Antagonists to corticotropin-releasing hormone and repeated parenteral infusion of TRH may have antidepressant activity when given during several weeks [Wong, 2000: Proc Natl Acad Sci USA 97:325-330; Arborelius et al., 1999: J Endocrinol 160:1-12; Callahan et al., 1997: Biol Psychiatry 41:264-272]. Manipulations of the sleep system through sleep deprivation can ameliorate depression [Szuba et al., 1994: Psychiatry Res 51:283-295; see Wirz-Justice et al., 1999: Biol Psychiatry 46:445-453 for review]. Sleep deprivation has been shown in more than three dozen studies published in the last three decades to produce marked, acute antidepressant effects in the majority of depressed individuals [Wirz-Justice, et al., 1999: Biol Psychiatry 46:445-453]. Thus, examination of the effects the two nonpharmacologic treatments, electroconvulsive therapy (ECT) and transcranial magnetic stimulation (TMS), produce in these physiologic systems may help elucidate their mechanisms of action, while enhancing understanding of the neurobiology of depressive illness. We will review these physiologic changes associated with depression, the effects that manipulations of these systems can have on depressive disorders, and then describe the effects the two techniques that can stimulate the human brain in vivo, ECT and TMS, exert on these systems.

Arousal↗

Role of nonspecific cytotoxic cells in the induction of programmed cell death of pathogenic protozoans: participation of the Fas ligand-Fas receptor system.

Numerous different species of parasites and pathogenic microorganisms produce programmed cell death (PCD) and apoptosis in eukaryotic targets. How ever, only a few studies have demonstrated that effector cells, cytokines, growth factors, or soluble apoptosis-inducing factors are capable of initiating apoptosis in protozoan parasites. Certain Tetrahymena spp. in teleosts are opportunistic pathogens. In the present study these pathogenic protozoans were developed as a model system to describe the potential role of the Fas ligand (FasL)-Fas receptor (FasR) system as a means of innate immunity in teleosts. Nonspecific cytotoxic cells (NCC) constitutively express soluble FasL (sFasL). Binding of the antigen receptor (i.e., NCCRP-1) on NCC to target cells caused the release of sFasL into the milieu. The presence of functional sFasL in these supernatants was determined by Western blot analysis and by demonstrating the lysis of FasR(+) HL-60 but not IM-9 (FasR(-)) targets. Soluble FasL containing supernatants generated by tumor cell-activated NCC also produced a reduction in 2 N DNA (i.e., DNA hypoploidy) of T. furgasoni. The induction of DNA hypoploidy by NCC supernatants could be neutralized by adsorption of the supernatants with anti-FasL antibody (but not with an isotype control). Experiments were next done to determine the expression of FasR on Tetrahymena and study the effects of anti-FasR monoclonal crosslinkage and treatment with soluble human recombinant FasL (huFasL) on initiation of PCD in Tetrahymena. Cell cycle analysis revealed that both crosslinkage and soluble huFasL binding to Tetrahymena produced DNA hypoploidy. The reduction in diploid DNA was confirmed by observing oligonucleosome fragmentation (DNA laddering) following anti-FasR treatment. Additional evidence for FasR expression on Tetrahymena was obtained using fluorescence microscopy and flow cytometry. Both methods showed that all Tetrahymena examined (three species consisting of four isolates) expressed membrane FasR. These studies demonstrated the potential of the FasL-FasR system in teleosts for initiation of antiparasite innate immunity. Effector NCC may initiate PCD of Tetrahymena that express a FasR-like protein. Induction of apoptosis may be a major mechanism of homeostatic control of protozoan parasite infestations/infections.

Animals↗

In vivo activation of tilapia nonspecific cytotoxic cells by Streptococcus iniae and amplification with apoptosis regulatory factor(s).

An important component of immediate innate responses of tilapia to stress is the release within minutes of soluble cytokine-like substances into the peripheral circulation. These cytokine-like stress factors bind nonspecific cytotoxic cells (NCC) and produce 3-4-fold increased cytotoxicity. In the present study, the in vivo responses of tilapia NCC following injection with different isolates of intact killed Streptococcus iniae was investigated. Activated cytotoxicity of NCC in the peripheral blood (PB) was produced by increased specific activity of resident cells rather than increased numbers. Tilapia injected intravenously (i.v.) with killed S. iniae produced different cytotoxicity responses compared to fish injected intraperitoneally (i.p.). In the spleen (S) and anterior kidney (AK), there was no correlation between S. iniae isolate and cytotoxicity response at 4, 8 or 24 h following i.p. injection. The NCC response following i.v. injection of killed bacteria was different. Within minutes following i.v. injection, NCC cytotoxicity from the PB increased 100% compared to naive controls. The existence of subsets of differentiated NCC in the PB was suggested because i.v. injection had no amplification effects on NCC from the AK or S. Likewise, NCC from the PB only appeared to exhibit a degree of antigen specificity. S. iniae strain #173 produced activation of cytotoxicity compared to isolates #164 and ATCC. Evidence for soluble factor (cytokine?) involvement in increased cytotoxicity was obtained by passive activation of NCC with serum from #173 (i.v.) injected fish. Incubation of this serum with control (naïve) NCC produced large increases in the cytotoxicity of labelled HL-60 target cells. Similarly obtained serum from fish injected with ATCC and #164 isolates had no amplification activity. Studies were also performed to study the mechanism(s) of passive activation. Flow cytometric analysis revealed that NCC from the S, AK and PB constitutively expressed cytosolic (not membrane) FasL. Stress serum treated NCC obtained from the peripheral blood produced an increase in the expression of FasL, CAS and FADD by Western blot examination. These data indicated that cytokine like factors in the serum of stressed tilapia activate increased NCC cytotoxicity (possibly) by stimulating the expression of proteins involved in activation of programmed cell death.

Animals↗

Use of sequence-specific antisense oligodeoxynucleotides to determine the protozoan parasite antigen recognized by nonspecific cytotoxic cells.

The antigen on the protozoan parasite Tetrahymena pyriformis recognized by catfish nonspecific cytotoxic cells (NCC) is a 46- to 48-kDa protein referred to as NKTag. The complete cDNA-derived amino acid sequence of NKTag has been obtained. The antigenic determinant of NKTag corresponding to the NCC binding site has been determined with synthetic peptides in target cell competition experiments. To more directly characterize the mechanism of parasite:effector cell interaction, we applied NKTag sequence-specific antisense oligodeoxynucleotides to Tetrahymena in vitro. NKTag mRNA translation by Tetrahymena was blocked by specific antisense (AS) oligodeoxynucleotides. 5'-3' sense (S) oligodeoxynucleotide sequences were synthesized corresponding to the first 17 N-terminal amino acids of NKTag (in addition to -2 untranslated codons plus the start codon). Complimentary AS oligodeoxynucleotides were likewise synthesized. To determine the optimum in vitro conditions for AS treatment, we tested parasites at various phases of their growth cycle for the effects of a single AS treatment. At 9 h post-AS treatment (during the linear phase of the growth curve), maximum reduction in membrane expression of NKTag was observed. Eighty-five percent of Tetrahymena were positive for expression of NKTag at 0 time post-AS treatment versus 13% positive at 9 h. Membrane expression of AS-treated parasites returned to normal levels by 24 h post-treatment. In cold target inhibition experiments, the reduced NKTag expression by Tetrahymena at 9 h AS treatment was confirmed by observing a complete inability (compared with S-treated parasites) to compete with IM-9 cells for binding with NCC. These data demonstrated a unique experimental in vitro system to define the antigen determinant on target cells responsible for recognition by cytotoxic effector cells that participate in innate immune responses.

Amino Acid Sequence↗

Regulation of innate immunity in tilapia: activation of nonspecific cytotoxic cells by cytokine-like factors.

Exposure of tilapia (Oreochromis niloticus) to water temperatures of 10-15 degrees C for 3-5 min produces physiological stress responses characterized by immediate phenotypic and immunological changes. In the present study, this general stress response was utilized as a model system to study innate immunity mediated by soluble factors and cytotoxic cells. Acute innate cytotoxic responses of nonspecific cytotoxic cells (NCC) in the peripheral blood (PBL), anterior kidney (AK) and spleen (SPL) were measured. Following temperature stress, the levels of NCC activity depended on the presence of soluble factors and on the cell compartments from which the NCC were obtained. NCC from PBL of stressed tilapia had 30x or greater cytotoxic activity compared to nonstressed PBLs from controls. NCC activity from the AK and SPL of stressed tilapia was lower than controls. Flow cytometric analysis of NCC in each tissue showed that increased cytotoxicity was not produced by increased numbers of NCC. To determine the mechanism of amplification of cytotoxicity, NCC from nonstressed tilapia were passively treated with serum from temperature stressed tilapia. Serum containing the "stress activated serum factor" (SASF) passively increased naive NCC cytotoxicity (from PBL) 3-4 fold. The cytotoxic cell response was inhibited by addition of anti-NCC monoclonal antibody 5C6. These data indicated that NCC are (at least one of) the target cells for SASF. SASF required only 15 min pre-incubation with naive NCC to activate cytotoxicity. Activation was nonreversible and concentration dependent. Pretreatment of NCC with SASF reduced the assay time required to amplify target cell cytotoxicity from 12-24 h to 6 h. SASF amplification of NCC cytotoxicity was not restricted by different histological types of target cells. Determination of select physical/chemical properties of SASF revealed: complete heat inactivation of cytotoxicity amplification following 55 degrees C and 65 degrees C pretreatment; SASF was thermostable at room temperature to 45 degrees C for 15 min; and freeze-thaw treatment reduced but did not completely remove amplification activity. The molecular weight range of SASF activity was identified in a 50-100 kDa fraction obtained by differential dialysis. SASF appears to be a protein sensitive to trypsin digestion.

Animals↗

Methods for cell cycle analysis and detection of apoptosis of teleost cells.

Flow cytometric techniques have not been previously used on a routine basis to study teleost cell growth and development. In the present chapter, flow instrumentation and cell preparation protocols are given in order to provide evaluation criteria characteristic of different phases of the cell cycle. Flow cytometry is used as an analytical and diagnostic tool to measure DNA ploidy as well as to measure alterations in cell cycle profiles characteristic of random DNA fragmentation (necrosis) compared to patterned DNA cleavage (apoptosis). The types of information obtained by flow analysis include the visualization of cell subpopulations with differing DNA content. For each identified nuclei subpopulation, the parameters of population size, fractions of nuclei in each phase of the cell cycle and computation of DNA ratios can be discerned. Data are presented of ex vivo prepared teleost nonspecific cytotoxic cells (NCC) at resting phase compared to NCC undergoing DNA hypoploid changes characteristic of apoptosis. These cells are compared with a teleost tissue cultured cell line maintained under optimum cell growth conditions versus cells undergoing necrotic cellular pathology. Finally, the requirements for optimum flow analysis are described. Techniques including gating strategies, voltage and gain settings, discrimination options and data collection and interpretation are provided.

Animals↗