Contrasting effects of interferon-gamma and interleukin-4 on neopterin generation from human adherent monocytes. The production of neopterin is closely correlated with activation of cell-mediated immunity. Neopterin appears to be produced by human macrophages specifically stimulated with interferon-gamma (IFN-gamma). Interleukin-4 (IL-4), a B and T stimulatory factor, has recently been shown to inhibit monocyte/macrophage functions, including the ability to suppress monocyte-generated cytokines. In this report we confirmed previous studies that identified the monocyte/macrophage as the main producing cell among human blood cells and that secretion is stimulated by IFN-gamma and lipopolysaccharides (LPS). IL-4 inhibits the generation of neopterin from unstimulated monocytes. This inhibitory effect was dose dependent and occurred at concentrations lower than 0.01 ng/ml. However, IL-4 had only a minimal inhibitory effect on LPS-induced generation of neopterin and could not reverse IFN-gamma-induced neopterin secretion from adherent monocytes. Furthermore, we report that LPS induced IFN-gamma production in monocyte culture. This production is strongly inhibited by IL-4 treatment. These findings indicate that IL-4 can regulate the synthesis of neopterin by adherent blood mononuclear cells and provide further evidence that LPS-induced neopterin in macrophages may act by IFN-gamma-independent mechanisms.


Beta-endorphin inhibits interleukin-2 release and expression of interleukin-2 receptors in concanavalin A-stimulated splenic lymphocytes. beta-endorphin, when added at the same time as the mitogenic lectin concanavalin A to mouse BALB/c spleen lymphocytes, inhibits cell proliferation. The suppressive effect of beta-endorphin is not exercised through a cAMP-dependent mechanism and is also observed when splenic lymphocytes are stimulated with phytohemagglutinin (4 micrograms/ml), anti-CD3 monoclonal antibody, or the Ca2+ ionophore A23187 (250 nM) and phorbol 12-myristate 13-acetate (1 ng/ml). The inhibitory effect of beta-endorphin on lymphocyte proliferation is dose and time dependent: when beta-endorphin is added 20 h after Con A stimulation no suppression of lymphocyte proliferation is observed. beta-Endorphin inhibits, in a dose-dependent manner, the release of interleukin-2 in concanavalin A-stimulated splenic lymphocytes, measured 24 h after stimulation. beta-Endorphin also controls the appearance of interleukin-2 receptors in the plasma membrane, but does not regulate the expression of the c-myc protooncogene. These data indicate that beta-endorphin inhibits lymphocyte activation signal transmission, downstream the generation of the second messengers Ca2+ and diacylglycerol and the expression of the protooncogene c-myc, by blocking interleukin-2 release and interleukin-2 receptors expression. Once the cells are in the G1 stage, beta-endorphin is no longer able to block lymphocyte proliferation.


Tracer kinetic evidence for abnormal methyl metabolism in schizophrenia. The original transmethylation hypothesis of schizophrenia has evolved with time and experiment to the present concept that a defect in the methyl-carbon metabolic pathway may be causative in this illness. Various researchers have proposed that specific steps in the methyl-carbon pathway may be defective, and have presented evidence to support these possibilities. We have tested the general concept of the hypothesis by administering methionine labeled with 11C or 14C in the S-methyl carbon to patients with schizophrenia and to controls and measured the expiration of 11CO2 and 14CO2. We found that the rate and total expiration of labeled CO2 were three times less in the patients than in the controls, with no overlap of data points in the two groups. Specific steps in the methylcarbon pathway that might be defective and produce the results seen here are discussed in light of this and other researchers' findings.


Magnetic resonance imaging of the posterior fossa in autism. The brainstem-cerebellar circuitry has been implicated in the pathophysiology of autism for several decades. Recent magnetic resonance imaging (MRI) studies of the posterior fossa have reported various abnormalities, the most noteworthy of which has been selective hypoplasia of the neocerebellar vermis. However, these initial MRI studies are limited by problems in both subject and control selection. The present study was undertaken to further investigate these MRI findings and the role of the cerebellum in autism, taking into consideration these methodologic issues. Eighteen high-functioning autistic subjects were recruited and matched with 18 normal controls on the basis of age, gender, IQ, race and socioeconomic status (SES). The midsagittal areas of the cerebellar vermis, vermal lobes, and the fourth ventricle were measured on 3 mm contiguous magnetic resonance images. Mean areas and standard deviations were comparable for all regions of interest and no statistically significant between-group differences were found. These negative findings argue against theories of autism based on gross structural abnormalities of the cerebellum. Previous reports of posterior fossa abnormalities may be related to technical and methodological factors, based on comparison of extant literature and recently available normative data.


Lithium in combination with perphenazine: effect on plasma monoamine metabolites. The addition of lithium to perphenazine altered the pattern of plasma homovanillic acid (HVA) during the course of treatment for acute psychosis. In the perphenazine-treated group plasma HVA declined significantly by days 7-9 of treatment, whereas in the perphenazine-plus-lithium group plasma HVA tended to increase. The pattern for plasma methoxyhydroxyphenethyl-glycol (MHPG) was not significantly different for the two groups. The addition of lithium to a neuroleptic may enhance the metabolism of dopamine.


Sleep EEG effects of 3,4-methylenedioxyethamphetamine (MDE; "eve") in healthy volunteers. 3,4-methylenedioxyethamphetamine (MDE; "Eve") exerts similar psychotropic effects in humans as 3,4-methylenedioxymethamphetamine (MDMA; "Ecstasy") and is less toxic in animal studies. We conducted a double-blind, placebo-controlled, cross-over sleep electroencephalogram (EEG) study with healthy volunteers. One hundred forty milligrams of MDE or placebo were administered PO in six subjects at 11 PM. Sleep EEG was registered from 11 PM-7 AM the next morning. All subjects had a normal sleep onset latency. They all awoke 60 to 120 min after administration of MDE and stayed awake for at least 150 min (total sleep time, TST MDE < placebo and intermittent time awake MDE > placebo: p < 0.001). After again falling asleep rapid eye movement (REM) sleep was totally suppressed (REM during time in bed, TIB MDE < placebo: p < 0.001). A cyclic alternation of relatively long periods of slow wave sleep (SWS) with periods of light sleep occurred in three subjects during the second part of the night (stage 4 in second part of night MDE > placebo: p = 0.16). The effects of MDE on sleep variables largely demonstrate the stimulant, amphetamine-like properties of MDE.


Late cognitive event-related potentials in adult Down's syndrome. Event-related electroencephalogram (EEG) potentials (ERPs) using two different tasks were measured in 14 adults with Down's syndrome (DS; mean age 32 years) without clinically detectable cognitive decline. Two groups, young healthy (YH) and old healthy (OH) adults, served as controls. In the oddball task, DS had prolonged N1 and earlier P2 latencies than the control groups. P3 latency was delayed in comparison to YH. In the PushWait task, P3 latency was later in DS than in YH and OH. In both tasks, DS showed a marked amplitude shift towards positivity overlapping the N1-P2 complex and seemingly also P3: The P3 amplitude evoked by target tones and by "Push" was shifted towards anterior sites resulting in a Cz maximum. Changes of the N1 latency and amplitude in DS may be related to enhanced arousal during stimulus processing, indicating a possible defect of central inhibitory mechanisms. The study suggests that differentiated ERP procedures provide information on adult DS cognition exceeding those given by mere P3 latency measurements. Such procedures may be useful in the evaluation of the cognitive decline due to precocious aging or Alzheimer-type dementia in DS.


Heightened motor activity level in male offspring of substance abusing fathers. Heightened activity level has been implicated in the liability for substance abuse, but no prior research has directly examined motor activity in a sample of prepubertal boys at high-risk for substance abuse. The magnitude of behavioral activity of 10-12 year-old sons of substance abusing fathers (SA +) (n = 42) and controls (SA -) (n = 60) was assessed using a microprocessor-based activity monitor during tasks that demanded significant concentration, effort, and constraint on motor activity, and when no such demands were present. Psychiatric status, measures of temperament traits, and measures of internalizing and externalizing behaviors were also obtained on the boys. Although the groups did not differ during the lower demand task, SA + boys showed significantly greater motor activity than SA - boys during the tasks that required concerted effort, attention, and behavioral suppression. Multiple regression analysis indicated that under the low-demand condition, the presence of an anxiety disorder in the boy was the only significant predictor of activity level. However, under the conditions that demanded effort, concerted attention and behavioral suppression, SA + group membership, and having low rhythmicity (as a temperament trait) predicted heightened activity. Statistically controlling for rhythmicity, the SA + boys were estimated to have about 24% higher activity than control boys. The results suggest that heightened motor activity may be associated with susceptibility to substance abuse.


The relationship between onsets of depression and sudden drops in solar irradiation. In a retrospective single-case study of a patient (L.S., the first author), who suffers from recurrent depression, we compared the onsets of his depression with meteorological data on light intensity, independently obtained, for the years 1975-1988. During those years there were 24 onsets or worsening of an existing depressive episode. Eleven of these occurred in the months September-December. Seven of these 11 autumn/winter onsets and 12 of the remaining 13 non-autumn/winter onsets coincided with a period of unusually low natural light intensity. Thus, 19 of 24 onsets (79%) were associated with falling light intensity, regardless of season. Some episodes that were not related to falling light intensity were associated with life events. For the years prior to the patient's use of light therapy, there was a statistically significant relationship between falling light and onset of depression. This supports the suggestion that depressive episodes (in certain individuals) may be triggered by unusually dull weather, regardless of season.


Metalloproteinase activities expressed during development and maturation of the rat prostatic complex and seminal vesicles. The objective of this study was to characterize proteinase activities expressed during development and maturation of the prostate gland and seminal vesicles of the rat by using gelatin-and casein-containing SDS polyacrylamide gel zymography. The prostatic complexes of 2- and 10-day-old animals and the individual lobes of the prostate (ventral, dorsolateral, and anterior [coagulating gland]) and the seminal vesicles of 15-day-old animals expressed prominent gelatinolytic activities of approximately 64, 71, and 76 kDa. These activities had properties of metalloproteinases; i.e., they were stimulated by Ca2+ and inhibited by EDTA and EGTA. They were greatly diminished by 52 days of age (immediately postpuberty) and were not detected in the dorsal lobe of the adult. Less active gelatinolytic proteinases with molecular masses of approximately 34 and 43 kDa were expressed in the developing prostatic complexes and individual lobes and seminal vesicles, but they were not detected in postpubertal animals. Weak gelatinolytic activities of 82, 85, and 89 kDa were found in the prostatic complexes; these activities were greatly diminished in all prostate lobes with sexual maturation but were expressed in the seminal vesicles at all ages. A large-molecular-mass Ca(2+)-independent proteinase of 130 kDa or greater was first detected in the dorsolateral prostate at 21 days of age. This activity was expressed in both the lateral and dorsal lobes of the adult but was greater in the lateral lobe. Proteinase activities of about 22 and 26 kDa that were not stimulated by Ca2+ were detected in the ventral prostate at 15 days of age by means of both gelatin and casein gels.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparison of norepinephrine and serotonin vasoconstriction of the rat isolated testicular subcapsular artery at physiological and elevated transmural pressures. This laboratory has previously described an in vitro preparation showing that the isolated testicular subcapsular artery of the adult rat has a novel triphasic transmural pressure-diameter myogenic response curve consisting of vasodilatation from 20 to 40 mm Hg, vasoconstriction from 40 to 100 mm Hg, and vasodilatation from 100 to 180 mm Hg, suggesting that the myogenic response of this artery between 40 and 100 mm Hg may have an important role in the autoregulation of the testicular blood supply. In the present studies, a 10-mm length of the adult rat isolated testicular subcapsular artery was cannulated and pressurized by an adjustable-height reservoir. External and internal arterial diameters were measured by a digital filar micrometer eyepiece. Dose-response curves for norepinephrine and serotonin were generated in a double-bath artery chamber at transmural pressures of 70 and 140 mm Hg, using half of the same artery for each pressure. Norepinephrine (3 x 10(-8) to 1 x 10(-5) M) produced a dose-dependent vasoconstriction at 70 mm Hg, with the highest dose causing a 31.4% decrease in lumen cross-sectional area (p < 0.05). Serotonin (3 x 10(-8) to 1 x 10(-6) M) produced a stronger dose-dependent vasoconstriction at 70 mm Hg, with the highest dose causing a 72.7% decrease in lumen cross-sectional area (p < 0.05). In marked contrast, the same concentration of norepinephrine and serotonin were found to have no statistically significant effect on the lumen cross-sectional area of the isolated testicular subcapsular artery at a transmural pressure of 140 mm Hg.(ABSTRACT TRUNCATED AT 250 WORDS)


Mature domestic cat oocyte does not express a cortical granule-free domain. A premature release of cortical granules (CG), found in the cortex of unfertilized oocytes, and the resulting formation of a CG-free domain (CGFD) over the metaphase II spindle are associated with nuclear maturation in the hamster and mouse. The objectives of our study were to characterize and compare the distribution of CG in immature, in vitro-matured, and in vivo-matured domestic cat oocytes while determining if a CGFD is formed that may be useful as a marker for stage and normalcy of oocyte maturation. Immature, intrafollicular oocytes were collected from ovaries obtained from local veterinary clinics, and a portion of these oocytes were matured in vitro. In vivo-matured, metaphase II oocytes were flushed from the oviducts of gonadotropin-treated, ovariohysterectomized cats. CG were visualized by Lens culinaris agglutinin-biotin/Texas red-strepavidin fluorescence, routine transmission electron microscopy, and Lens culinaris agglutinin-biotin/gold-labeled strepavidin transmission electron microscopy. No CGFD was detected in any domestic cat oocyte. Immature, in vitro-matured, and in vivo-matured oocytes had similar, uniform distributions of CG throughout the entire cortical region as measured by fluorescence microscopy. In vivo-matured oocytes were further examined by transmission electron microscopy, which confirmed the lack of a CGFD. All oocytes contained CG having a mean diameter of 0.28 +/- 0.03 micron/granule and a mean density of 51.5 +/- 13.0 CG/100 microns of plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)


Competition between zonae pellucidae and a proteinase inhibitor for sperm binding. Murine sperm bind a proteinase inhibitor of seminal vesicle origin at ejaculation. The inhibitor binds in the acrosomal region of the sperm head and is removed during in utero or in vitro incubation. Adding inhibitor to sperm reduces their ability to bind zonae, while adding the purified inhibitor binding site to cumulus-free, zona-intact oocytes reduces the ability of the oocytes to bind sperm. Immuno-aggregation of the inhibitor binding site results in exocytosis of the acrosome. These observations suggest that the inhibitor binding site may participate in zona binding and the acrosome reaction. If the inhibitor binding site binds both the zona and the seminal inhibitor, then these components should compete with each other for that site on the sperm. We show that purified seminal inhibitor, as well as other proteinase inhibitors, block zona-induced acrosome reactions. Likewise, zona glycopeptides block inhibitor/anti-inhibitor-induced acrosome reactions in a concentration-dependent fashion. The inhibitor/anti-inhibitor-induced acrosome reaction is sensitive to pertussis toxin and proteinase inhibitor and thus is similar to zona-induced reactions. These findings support the suggestion that the trypsin inhibitor binding site on the head of the sperm functions to insure sperm-zona binding and induction of the acrosome reaction.


Intrauterine position effects on steroid metabolism and steroid receptors of reproductive organs in male mice. Mice differ in their adult reproductive characteristics as a function of whether they developed in utero between two male fetuses (2M males), which have higher testosterone levels, or between two female fetuses (0M males), which have higher estradiol levels. The present study was designed to further characterize biochemical parameters of 2M and 0M adult male mice. Activities of testicular steroidogenic enzymes, namely delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase, 17 alpha-hydroxylase, and C17,20-lyase (C21SCC P450), were measured by means of radiometric assays and HPLC fractionation of substrate and products. Activity of 5 alpha-reductase in both seminal vesicle and prostate was measured by similar techniques. Estrogen and androgen receptor concentrations, which indicate capacity to respond to steroid hormones, were also examined in the accessory sex organs. For both seminal vesicle and prostate, 5 alpha-reductase activities were approximately 60% greater in 2M males than in 0M males, indicating greater capacity to form dihydrotestosterone from testosterone in organs from 2M mice. No significant differences were found in testicular steroidogenic enzymes between 2M and 0M animals, whereas the trend for all three activities was higher for 2M males than for 0M males. While no differences were found in estrogen receptor concentrations, 0M prostates had three times the concentration of androgen receptors (occupied receptors) compared to 2M prostates. Our findings suggest that intrauterine fetal position exerts a significant influence on subsequent adult androgen metabolism and androgen responsiveness in reproductive organs of adult male mice.


Gonadotropin-independent mechanisms participate in ovarian responses to realimentation in feed-restricted prepubertal gilts. Short-term feed restriction in prepubertal gilts suppresses episodic LH secretion in the absence of changes in body weight or composition. To assess non-gonadotropin-mediated effects of realimentation at the ovarian level, 52 gilts were assigned to six treatments after 7 days (Days 1-7) of maintenance feeding (approximately 30% ad libitum). Groups R12 and R9 were maintenance-fed Days 8-12 or Days 8-9, respectively; A12 and A9 were fed to appetite Days 8-12 or Days 8-9, respectively. Groups R9P and A9P were fed as groups R9 and A9 were but received 750 IU eCG at 1500 h on Day 8. Groups R12 and A12 were ovariectomized at 1500 h on Day 12, and all other groups were ovariectomized at 1500 h on Day 9. All gilts received oral progestogen (15 mg allyl trenbolone) from Day 1 to ovariectomy, to antagonize the usual increases in endogenous gonadotropins that follow realimentation. Blood samples were obtained at 10-min intervals during selected windows during the experiment. Ovarian follicles were analyzed for development and steroidogenesis, and plasma samples were analyzed by RIA to determine concentrations of LH, FSH, insulin, and insulin-like growth factor-1 (IGF-1). Allyl trenbolone abolished pulsatile LH secretion, and realimentation did not stimulate LH or FSH secretion, with the exception of FSH secretion on Day 8 in A9 gilts. Postprandial insulin concentrations on Day 9 were greater after feeding to appetite (A9, A9P, and A12) than after feed restriction (R9, R9P, and R12). Pre- and postprandial IGF-1 concentrations were higher in re-fed gilts on Day 9 (A9 and A12) and Day 12 (A12) than in feed-restricted gilts. Follicular diameter, fluid volume, and basal granulosa cell estradiol synthesis per follicle were greater in A12 gilts than in R12 gilts, although there was no difference between A9 and R9 gilts. There was no effect of realimentation on follicular fluid concentrations of estradiol or testosterone, or on androgen-driven granulosa cell estradiol synthesis. Treatment with eCG increased follicular diameter, fluid volume, basal and androgen-driven estradiol synthesis, and fluid estradiol concentrations without interaction with feeding level. In conclusion, in the absence of LH elevations, realimentation over 5 days exerts effects at the ovary, increasing follicular growth and estradiol synthesis. These effects may be mediated by insulin, IGF-1, or unmeasured growth factors and would be expected to synergize with increases in endogenous gonadotropin that follow realimentation.


Immunoneutralization of inhibin suppresses reproduction in female mink. This study determined whether immunoneutralization of inhibin affected gonadotropin secretion, embryo development, and ovarian function in mink. Adult female mink (n = 10) were immunized with bovine inhibin alpha 1-26 gly-tyr (bINH, 100 micrograms) conjugated to human alpha globulins (HAG), or with HAG alone (n = 10, controls), mixed with Freund's complete adjuvant. A series of five boosters containing bINH or HAG were then administered during a 2-yr period. Titers of bINH antibodies and serum concentrations of gonadotropins were determined for each breeding season in 1990 and 1991. Each year after whelping, we determined gestation length; sex, number, and weight of live and dead kits per litter at birth; and number and weight of kits per litter 3 wk after whelping. Results were pooled for statistical analysis. Bovine INH antibody titers (percent 125I-bINH bound to serum diluted 1:8000) were 53 +/- 3% vs. 2 +/- 0.6%, and serum concentrations of FSH were higher (p < 0.05) in bINH-immunized mink compared with controls (144 +/- 23 vs. 67 +/- 12 ng/ml). However, number (3.8 +/- 0.2 vs. 5 +/- 0.4) and weight (8 +/- 0.3 vs. 9.7 +/- 0.4 g) of kits per litter at birth and number of kits per litter alive 3 wk after birth (2.9 +/- 0.5 vs. 4.7 +/- 0.4) were lower (p < 0.05) in bINH-immunized mothers compared with controls. During the nonbreeding season in 1991, a single injection of hCG (100 IU) was administered to bINH-immunized and control mink; 24 h later blood was sampled, and organ weights were determined.(ABSTRACT TRUNCATED AT 250 WORDS)


Role of human sperm phospholipase A2 in fertilization: effects of a novel inhibitor of phospholipase A2 activity on membrane perturbations and oocyte penetration. Phospholipase A2 was isolated from human sperm and its potential role in the membrane fusion events of fertilization was examined. Highly purified enzyme hydrolyzed the phospholipids of [1-14C]oleate-labeled Escherichia coli optimally at neutral to alkaline pH with 5 mM CaCl2 and 150 mM NaCl (specific activity = 20 mumol/min/mg). Activity was inhibited in a dose-dependent manner by an oligomer of prostaglandin B1 (IC50 = 1.5 microM) reported to inhibit human phospholipases A2 in vitro and in situ. Sperm phospholipase A2 injected into mouse foot pad induced a dose-dependent edema that was inhibited by oral administration of prostaglandin Bx (IC50 < or = 10 mg/kg) or by pretreatment of the enzyme with 4-bromophenacyl bromide. Human sperm phospholipase A2 (10 micrograms) induced fusion of phosphatidylserine vesicles in the presence of 1 mM calcium chloride by approximately 80% (+/- 10%) as determined by monitoring turbidity (O.D.400) and efficiency of fluorescence resonance energy transfer. This enzyme-induced fusion was accompanied by phospholipid hydrolysis, and both fusion and phospholipid degradation were inhibited by more than 60% when enzyme was preincubated with 5 microM prostaglandin Bx. Sperm penetration of zona pellucida-free hamster oocytes was inhibited in a dose-dependent fashion when sperm were incubated with prostaglandin Bx (IC50 approximately 15 microM) during capacitation; sperm motility was not affected by this treatment. Capacitation in the presence of prostaglandin Bx had little to no effect on the in vitro acrosome reaction. These results suggest that sperm phospholipase A2 and its modulators may contribute to membrane fusion events in mammalian fertilization.


Bovine luteinizing hormone (LH) isoforms and amounts of messenger ribonucleic acid for alpha- and LH beta-subunits in pituitaries of cows immunized against LH-releasing hormone. Ovariectomized beef cows were actively immunized against LHRH to test the hypothesis that decreased stimulation of gonadotropes would alter the distribution of LH isoforms and amounts of mRNA for subunits of LH in the pituitary. Eight long-term (3 yr) ovariectomized beef cows were randomly assigned to one of two treatments: immunization against LHRH conjugated to human serum globulin (n = 4) and nonimmunization (control, n = 4). Mean concentration of serum LH in cows immunized against LHRH (1.0 +/- .83 ng/ml) was less (p = 0.01) than in control cows (5.0 +/- 0.83 ng/ml). Amounts of alpha- (p = 0.13) and LH beta-subunit (p = 0.10) mRNA tended to be reduced in cows immunized against LHRH compared to control cows. However, weight of the anterior pituitary and concentrations of LH in this gland did not differ (p = 0.90) among cows from the two groups. Pituitary extracts were chromatofocused on pH 10.5-7.0 gradients, and concentrations of LH in eluent fractions were determined by RIA. Extracts of all pituitaries resolved into nine isoforms (designated A through H and Z beginning with the most basic form). Only isoform F (mid-alkaline elution, pH = 8.8) was influenced by treatment (p = 0.05). Cows immunized against LHRH had a greater relative amount of isoform F (42.1 +/- 1.4%) than controls (37.2 +/- 1.4%). In summary, immunization of cows against LHRH altered 1) circulating concentrations of LH, 2) amounts of mRNA for the subunits of LH, and 3) distribution of intrapituitary LH isoforms without changing the concentrations of LH in the anterior pituitary.


Compensatory development in preimplantation mouse embryos derived from delayed mating. Rodent embryos resulting from delayed mating grow relatively faster than those resulting from normal mating. To evaluate this phenomenon quantitatively, in the present study we compared the number of cells at the preimplantation stage of mouse embryos derived from normal mating and those derived from delayed mating (3 and 6 h after ovulation). The mean cell numbers (45.4 and 43.0 for delayed mating at 3 and 6 h postovulation, respectively) of embryos at 77 h postcoitus (hpc) in the delayed mating groups were greater than that (38.4) of the normal mating group. Further, when the mean cell numbers (38.8 and 38.5) in the delayed mating groups were counted at 74 hpc, they were almost equal to that of the normal mating group at 77 hpc. The study demonstrated that preimplantation mouse embryos derived from delayed mating progress more rapidly than their normally mated counterparts. However, a 3-h advance in development seems to be the limit of this increased rate of growth, even when the time interval from ovulation to mating is longer than 3 h. The mechanism(s) of this interesting compensatory phenomenon should be investigated.


Initiation and maintenance of in vitro decidualization are independent of hormonal sensitization in vivo. The effects of in vivo hormonal sensitization on the competence of uterine stromal (US) cells to decidualize in vitro were assessed. In vitro differentiation of uterine stroma isolated from Day 4 pregnant rats, sensitized to respond to a decidual stimulus, was compared to that in nonsensitized immature, castrated or cycling rats. The initiation of in vitro decidualization--as monitored by the expression of the decidual markers desmin and laminin in rat US cells--was independent of the hormonal status of the animal from which the cells were isolated and occurred in the absence of serum in the medium. Differentiation was accelerated in high-density cultures where contact inhibition suppressed proliferation and decreased the extent of cell growth. The extent to which in vitro decidualization imitates in vivo stromal cell differentiation was assessed by comparing decidualization in the rabbit, a species with only a limited decidual cell response, and in the rat. US cells isolated from nonpregnant rabbits differentiated in vitro by expressing laminin, but not desmin. Indirect immunofluorescence of frozen uterine sections from pregnant and nonpregnant rabbits validated in vitro differentiation as a faithful reflection of the in vivo program of decidualization. Although the program of US cell differentiation may vary between the species, initiation of differentiation in vitro appeared to be independent of hormonal preparation in vivo for both the species examined.


Ontogeny and cellular localization of 125I-labeled basic fibroblast growth factor and 125I-labeled epidermal growth factor binding sites in ovaries from bovine fetuses and neonatal calves. We have recently reported that although specific 125I-FSH receptors are present in granulosa cells from primary and secondary follicles, gonadotropin responsiveness is very low in ovaries from bovine fetuses, which consist mainly of preantral follicles with few early antral follicles. It is well established that a number of polypeptide growth factors show pronounced mitogenic effects on follicular cells. Therefore, we have compared autoradiographically the ontogeny and cellular localization of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) binding activities to assess their possible involvement in the regulation of early follicular growth in fetuses and neonatal calves. Follicular growth was initiated around Day 180 of gestation in fetuses. 125I-bFGF binding values were high in granulosa cells from preantral follicles (mean +/- SEM, 7.8 +/- 1.1-9.8 +/- 0.7 grains/cell, 0.05-0.15-mm diam.) but decreased in early antral follicles (0.16-3.0 mm) to a constant level (5.7 +/- 1.2 grains/cell). Specific 125I-EGF binding values were low in preantral follicles but showed a 2.5- and 5.0-fold increase in both granulosa cells and the theca interna from antral I (0.16-0.5 mm) and antral II follicles (0.6-3.0 mm), respectively. In atretic follicles, 125I-bFGF specific binding values were high (10.4 +/- 0.8 grains/cell), whereas 125I-EGF binding levels were significantly reduced or absent. None of the radioligands tested bound significantly to primordial follicles. There was no age-related difference in any ligand binding to follicles of comparable size. These results provide novel evidence that bFGF, a potent mitogen, is involved in the regulation of granulosa cell function as early as the preantral stage in cattle.(ABSTRACT TRUNCATED AT 250 WORDS)


Ontogeny and cellular localization of 125I-labeled insulin-like growth factor-I, 125I-labeled follicle-stimulating hormone, and 125I-labeled human chorionic gonadotropin binding sites in ovaries from bovine fetuses and neonatal calves. In a previous study we reported that ovaries from bovine fetuses, which consist mainly of preantral follicles with few antral follicles, are weakly responsive to gonadotropins (FSH and LH). Insulin-like growth factor-I (IGF-I) is known to enhance gonadotropin responsiveness in vitro, but there is a lack of consistent data on the involvement of IGF-I, FSH, and LH during early stages of folliculogenesis in cattle. In the study reported here, we assessed autoradiographically the ontogeny of 125I-gonadotropin and 125I-IGF-I binding activities during preantral and early antral stages in cattle. Follicular growth was initiated around Day 180 of gestation in fetuses. The density of 125I-FSH binding was high in granulosa cells from primary (mean +/- SEM 10.5 +/- 0.7 grains/cell, 0.05-mm diam.) and secondary follicles (10.8 +/- 0.8 to 13.6 +/- 1.2 grains/cell, 0.06-0.15 mm) but increased significantly (p < 0.05) in early antral follicles (18.2 +/- 1.1 grains/cell, 0.16-3.0 mm). Specific 125I-IGF-I binding levels were low in granulosa cells from preantral follicles, averaging 2.5 +/- 0.6-3.1 +/- 0.9 grains/cell. However, after antrum formation, the density of 125I-IGF-I binding increased significantly (p < 0.05) with follicular diameter in granulosa cells and was 5.7 +/- 0.7 and 9.1 +/- 0.6 grains/cell for antral I (0.16-0.5 mm) and antral II (0.6-3.0 mm) follicles, respectively. 125I-FSH and 125I-IGF-I binding densities were low in theca cells from preantral and early antral follicles as well as in the interstitial tissue and granulosa cells from atretic follicles.(ABSTRACT TRUNCATED AT 250 WORDS)


Regulation of the synthesis and secretion of transferrin and cyclic protein-2/cathepsin L by mature rat Sertoli cells in culture. While numerous studies have examined the response of immature rat Sertoli cells to specific hormones and growth factors, the regulation of mature cells in vitro has not been well examined because highly purified cells have been difficult to isolate. We now describe a detailed method for isolating Sertoli cells from mature (> 60 days of age) rats and generating primary cultures of these cells greater than 90% in purity. We demonstrate that cell density, hormones, and growth factors regulate the synthesis or secretion of two Sertoli cell products, transferrin and Cyclic Protein-2 (CP-2)/cathepsin L. Cell density modulated the response of mature Sertoli cells to some hormones; insulin (at 10 micrograms/ml) and epidermal growth factor (EGF) acted synergistically to stimulate transferrin synthesis by 80% when cells were cultured at a density of 1.65 x 10(5) cells/cm2 but had no effect on transferrin synthesis by cells cultured at 1.46 x 10(5) cells/cm2. A mixture of FSH, retinol, and testosterone increased transferrin synthesis by 30% at both cell densities, and this stimulation was independent of the effect of EGF and insulin. CP-2/cathepsin L synthesis was significantly stimulated by increased cell density. FSH, retinol, and testosterone also stimulated CP-2/cathepsin L synthesis by 30%; however, this stimulation just missed being statistically significant. Finally, we demonstrated that secretion of transferrin and CP-2 was reduced when cells were cultured in the presence of interleukin-1 alpha, a cytokine synthesized by Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Isolation and characterization of a 25-kilodalton protein from mouse testis: sequence homology with a phospholipid-binding protein. A 25-kDa epididymal secretory protein (MEP 9), isolated from mouse epididymal fluid, has recently been characterized in our laboratory [Rankin et al., Biol Reprod 1992; 46:747-766]. The polyclonal antibody raised against this protein was found to recognize a 25-kDa component in epididymal fluid and testicular extract. The 25-kDa testicular antigen (MTP) was purified by means of ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography; MTP was found to be similar to MEP 9 in several properties including molecular mass (25 kDa), isoelectric point (pI 6.0), and immunoreactivity when the proteins were resolved in the presence of SDS (one-dimensional and two-dimensional PAGE). However, when the proteins were resolved under non-denaturing conditions, MTP showed strong immunoreactivity while MEP 9 did not. This observation suggests that although the 25-kDa antigens from the epididymal fluid and testicular extract are quite similar, they may have different immunological conformations. When analyzed for amino acid composition and partial amino acid sequence, the testicular antigen showed substantial homology (> 80%) with a phosphatidylethanolamine-binding protein characterized from bovine brain. MTP also showed phosphatidylethanolamine-binding activity (Kd = 1.95 x 10(-5) M, Bmax = 1.86 nmol/micrograms MTP), suggesting that the mouse 25-kDa protein is a member of the phospholipid-binding protein family and may have a role in lipid metabolism during sperm maturation.


Immunolocalization of a 25-kilodalton protein in mouse testis and epididymis. We have recently observed that a polyclonal antibody raised against a mouse epididymal luminal fluid protein (MEP 9) recognizes a 25-kDa antigen in mouse testis and epididymis [Rankin et al., Biol Reprod 1992; 46:747-766]. This antigen was localized by light and electron microscopic immunohistochemistry. The immunoreactivity in the testis was found in the residual cytoplasm of the elongated spermatids, in the residual bodies, and in the cytoplasmic droplets of spermatozoa. In the epididymis, the epithelial principal cells were stained from the distal caput to the distal cauda. Immunogold labeling in the principal cells showed diffuse distribution without preferential accumulation in either the endocytic or the secretory apparatus of the cells. In the epididymal lumen, the immunoreactivity was restricted to the sperm cytoplasmic droplets. No membrane-specific labeling was observed in luminal spermatozoa, cytoplasmic droplets, or isolated sperm plasma membranes. Three weeks after hemicastration or severance of the efferent ducts, a normal distribution of the immunoreactive sites was found in the epididymis. Immunoreactivity, was also detected in the epididymal epithelium of immature mice as well as in that of XXSxr male mice having no spermatozoa in the epididymis. These results suggest that the immunoreactivity seen in the principal cells originates from synthesis rather than endocytosis of the testicular protein from disrupted cytoplasmic droplets. Furthermore, these results suggest that the 25-kDa protein is synthesized independently by both testis and epididymis.


Molecular analysis of the antigenicity and immunogenicity of recombinant zona pellucida antigens in a primate model. The studies reported here are the first to demonstrate that recombinant zona pellucida (ZP) proteins will elicit a humoral immune response that recognizes native ZP proteins. Three cDNAs encoding rabbit ZP protein antigens expressed in bacteria were used to immunize cynomolgus monkeys. Four groups of six monkeys each were immunized with bacterially expressed cro-beta-galactosidase recombinant proteins encoded by a full-length cDNA (rc55) encoding the 55-kDa rabbit ZP recombinant protein (rec55), two partial cDNAs (rc75a and rc75b) encoding two recombinant peptides (rec75a and rec75b) of the 75-kDa rabbit ZP protein, and the plasmid-encoded cro-beta-galactosidase control protein. Initial immunizations with these fusion proteins using the muramyl dipeptide adjuvant did not elicit significant levels of antibodies to native or recombinant ZP proteins. Further immunizations were therefore carried out using recombinant ZP proteins conjugated to either protein A or keyhole limpet hemocyanin. Antibodies were detected in the groups immunized with the rec55 and rec75a; however, no antibodies were generated against the rec75b protein. These antibodies have been characterized by two-dimensional PAGE immunoblotting and shown to recognize antigenic domains associated with two of the native rabbit ZP proteins. Reprobes of these immunoblots with sheep anti-total native rabbit ZP proteins, affinity-purified on pig ZP, further demonstrate that a fourth distinct rabbit ZP antigen may be present. The characterization of species-conserved antigenic domains of mammalian ZP proteins is important for studies of the functional regions of ZP proteins and is critical for the design of safe and effective contraceptive vaccines.


Changes in concentration of adenosine triphosphate and adenosine diphosphate in individual preimplantation sheep embryos. Concentrations of ATP and ADP were measured in 156 sheep embryos by means of an ultramicrofluorescence assay. Stages of preimplantation development measured included unfertilized oocytes through blastocyst-stage embryos. ATP concentrations remained constant through the 8-cell stage; then ATP decreased significantly (p < 0.025) at the morula stage and remained low through the blastocyst stage. ADP concentrations did not change throughout the embryonic stages measured. Decreased levels of ATP with constant levels of ADP caused the ATP:ADP ratio to decrease significantly (p < 0.025) between the 8-cell and morula stages. We suggest that the increase in glucose uptake by sheep embryos observed at the morula stage of development may be due, in part, to a decrease in the ATP:ADP ratio.


Endocrine and ovarian responses associated with the first-wave dominant follicle in cattle. To examine endocrine and biochemical differences between dominant and subordinate follicles and how the dominant follicle affects the hypothalamic-pituitary-ovarian axis in Holstein cows, the ovary bearing the dominant follicle was unilaterally removed on Day 5 (n = 8), 8 (n = 8), or 12 (n = 8) of synchronized estrous cycles. Follicular development was followed daily by ultrasonography from the day of detected estrus (Day 0) until 5 days after ovariectomy. Aromatase activity and steroid concentrations in first-wave dominant and subordinate follicles were measured. Intact dominant and subordinate follicles were cultured in 4 ml Minimum Essential Medium supplemented with 100 microCi 3H-leucine to evaluate de novo protein synthesis. Five days after unilateral ovariectomy, cows were resynchronized and the experiment was repeated. Follicular growth was characterized by the development of single large dominant follicles, which was associated with suppression of other follicles. Concentrations of estradiol-17 beta (E2) in follicular fluid and aromatase activity of follicular walls were higher in dominant follicles (438.9 +/- 45.5 ng/ml; 875.4 +/- 68.2 pg E2/follicle) compared to subordinate follicles (40.6 +/- 69.4 ng/ml; 99.4 +/- 104.2 pg E2/follicle). Aromatase activity in first-wave dominant follicles was higher at Days 5 (1147.1 +/- 118.1 pg E2/follicle) and 8 (1028.2 +/- 118.1 pg E2/follicle) compared to Day 12 (450.7 +/- 118.1 pg E2/follicle). Concentrations of E2 and androstenedione in first-wave dominant follicles were higher at Day 5 (983.2 +/- 78.2 and 89.5 +/- 15.7 ng/ml) compared to Days 8 (225.1 +/- 78.6 and 5.9 +/- 14.8 ng/ml) and 12 (108.5 +/- 78.6 and 13.0 +/- 14.8 ng/ml). Concentrations of progesterone in subordinate follicles increased linearly between Days 5 and 12 of the estrous cycle. Plasma concentrations of FSH increased from 17.9 +/- 1.4 to 32.5 +/- 1.4 ng/ml between 0 and 32 h following unilateral removal of the ovary with the first-wave dominant follicle. Increases in plasma FSH were associated with increased numbers of class 1 (3-4 mm) follicles in cows that were ovariectomized at Day 5 or 8 of the cycle. Unilateral ovariectomy had no effects on plasma concentrations of LH when a CL was present on the remaining ovary. First-wave dominant follicles incorporated more 3H-leucine into macromolecules and secreted high (90,000-120,000) and low (20,000-23,000) molecular weight proteins that were not as evident for subordinate follicles at Days 8 and 12.(ABSTRACT TRUNCATED AT 400 WORDS)


Pituitary response to repeated copulation and/or gonadotropin-releasing hormone administration in llamas and alpacas. The response of the pituitary gland and ovary to repeated copulatory periods and/or gonadotropin-releasing hormone (GnRH, i.v. 1000 micrograms) administration was determined in llamas and alpacas. Eighty adult females (41 llamas and 39 alpacas with ovulatory follicles) were divided into three general groups for each species as follows: copulation (one or two copulations at either 6- or 24-h intervals) GnRH treatment (one or two treatments at either 6- or 24-h intervals), and combined treatment (copulation followed by GnRH treatment, or GnRH followed by copulation at either 6- or 24-h intervals). An additional control (nontreated) group was composed of 4 llamas and 4 alpacas. The first copulation or treatment with GnRH provoked LH release sufficient to cause ovulation in most of the females (alpacas, 89%; llamas, 92%); urinary pregnanediol glucuronide values, used to verify ovulation, were significantly elevated 48 h after copulation and/or GnRH treatment. A second stimulus, copulation or GnRH, provoked no LH response with concentrations similar to those in nontreated controls and in females not ovulating. Llamas and alpacas thus were refractory to a second copulatory or GnRH stimulus with regard to LH release for up to 24 h following an initial ovulatory release of LH.


Biosynthesis and immunocytochemical localization of an estrogen-dependent glycoprotein and associated morphological alterations in the sheep ampulla oviduct. Published reports have shown that an M(r) 90,000-92,000 protein is released into the oviductal lumen of the sheep, during estrus at a time corresponding to ovulation and fertilization, where it associates with the embryo. The objectives of this study were (a) to determine whether estradiol-17 beta (E) alone or in combination with progesterone (P) induces the synthesis of the M(r) 90,000-92,000 protein from the ampulla and/or isthmus oviduct; (b) to monitor structural alterations in oviductal epithelial cells associated with the synthesis of this protein; and (c) to generate a polyclonal antiserum to the protein and use the antiserum to verify its cellular location and tissue specificity. Oviductal flushings and explant culture media were obtained from ovariectomized animals treated with E alone or with E plus P. The M(r) 90,000-92,000 protein was present in 3H-leucine- and 3H-glucosamine-labeled culture media of the ampulla (not isthmus) oviduct in animals treated with E alone or with E plus P. The glycoprotein was detected in gels of oviductal flushings obtained from animals treated only with E. A specific polyclonal antiserum to the protein was made and cross-reacted on Western blots of oviductal flushings from E-treated animals and ampulla (not isthmus) oviduct culture media from animals treated with E alone or with E plus P. The secretory apparatus of the epithelial cells of the ampulla oviduct matured and differentiated in response to E. Light and electron microscopic immunocytochemistry localized the M(r) 90,000-92,000 glycoprotein to secretory granules in the nonciliated cells of the ampulla oviduct. Immunoperoxidase reaction product was absent in tissue sections and Western blots of other reproductive and nonreproductive tract tissues obtained from steroid-treated animals. Therefore, the secretory cells of the ampulla oviduct of the sheep synthesize and release an E-induced, oviduct-derived M(r) 90,000-92,000 glycoprotein.


Fluctuations of lactoferrin protein and messenger ribonucleic acid in the reproductive tract of the mouse during the estrous cycle. The physiological role of lactoferrin (LF), the major estrogen-inducible protein in the murine uterus, is unclear; however, LF may be a useful marker for the study of estrogen action in the uterus. Thus, the expression of LF mRNA and the localization of the protein in genital tract tissues and secretions of female mice (6-8 wk old) at different stages of the estrous cycle were investigated. Uterine luminal fluid (ULF) was analyzed for LF by means of gel electrophoresis and Western blot techniques; LF mRNA and protein were identified in reproductive tract tissues through in situ hybridization and immunocytochemistry. At diestrus, the level of LF mRNA was low, and staining for the protein was very light in uterine epithelial cells; LF was undetectable in ULF. At proestrus, LF mRNA and protein increased in the uterine epithelium and LF was readily detectable in ULF. LF mRNA and protein reached the highest levels at estrus. At early metestrus as compared to estrus, LF mRNA and protein were detected in decreasing amounts in uterine epithelial cells; the protein was undetected in ULF. By late metestrus and diestrus, LF mRNA and protein returned to a low level, and the protein was undetectable in ULF. LF protein was also demonstrated by immunocytochemistry in the epithelium of the oviduct, cervix, and vagina. LF protein fluctuation similar to that observed in the uterus was seen in these tissues; however, the uterus demonstrated the most dramatic changes in the number of epithelial cells involved in LF production during the estrous cycle. In summary, LF mRNA and its expression in uterine epithelial cells of the mouse varied with the stage of the estrous cycle. These results, combined with previously reported findings that LF is a major constituent of mouse ULF under the influence of estrogen, suggest that LF may play an important role in normal reproductive processes.


Evaluation of new anti-infective drugs for the treatment of chancroid. Infectious Diseases Society of America and the Food and Drug Administration. Chancroid is a mucocutaneous infection caused by Haemophilus ducreyi that produces ulcerative lesions and enhances the efficiency of transmission of human immunodeficiency virus (HIV). Confirmation of infection by culture of H. ducreyi is essential in therapeutic trials. Minimal inhibitory concentrations of antibiotics for the isolate should be determined by agar dilution. Patients should be evaluated by appropriate laboratory tests for syphilis, infection with herpes simplex virus, gonorrhea, and (in North America) infection with Chlamydia trachomatis. The clinical history of the disease should be recorded and ulcers, buboes, and lymphadenitis mass described. Whenever possible, study participants also should be tested for HIV infection. Randomized, prospective, double-blind, active-control comparative clinical trials are preferred for evaluation of the safety and efficacy of new anti-infective drugs. Otherwise-healthy men and women should be enrolled in these studies. Patients with active syphilis or genital herpes should be excluded. Microbiological and clinical outcomes are paramount.


Evaluation of new anti-infective drugs for the treatment of vaginal infections. Infectious Diseases Society of America and the Food and Drug Administration. The three major vaginal infections are yeast vulvovaginitis, Trichomonas vaginalis vaginitis, and bacterial vaginosis. In terms of signs and symptoms, these disorders overlap substantially with one another and with other infections. Therefore, the diagnosis of candidiasis and trichomoniasis requires isolation of the responsible pathogen. For the diagnosis of bacterial vaginosis, all other potential causes of vaginal infection must be excluded and specified laboratory criteria must be met. Clinical trials must be carefully designed to control for coexisting pathogens, for potential efficacy of treatment against more than one microbe, and for variable end points used to define clinical response. Prospective, randomized, double-blind, active-control comparative studies are preferred. Follow-up evaluations 5-7 days and 4-6 weeks after the completion of therapy are required for the assessment of outcome. Laboratory studies of vaginal fluid (culture and/or microscopic examination) are paramount in the final appraisal of outcome.


Evaluation of new anti-infective drugs for the treatment of uncomplicated gonorrhea in adults and adolescents. Infectious Diseases Society of America and the Food and Drug Administration. Gonorrhea is among the most common sexually transmitted diseases. Treatment for uncomplicated gonorrhea should be efficacious in > or = 95% of cases. Because patients with gonococcal infections often have other sexually transmitted diseases concurrently, individuals enrolled in clinical trials of therapy for gonorrhea should also be evaluated for infection with Chlamydia trachomatis and for syphilis. Testing for other pathogens should be considered in light of the clinical presentation. The presence of gonococcal infection is defined by a positive culture of a specimen obtained from an appropriate mucosal site. Patients enrolled in clinical trials should be otherwise-healthy adults who agree to return for follow-up assessment. These patients should be stratified by gender and anatomic site of infection. The preferred study design is a prospective, randomized, double-blind, active-control comparison. In some circumstances, however, historical controls may suffice. The study drug must have an efficacy rate of > or = 95% in genital and rectal infections. Microbiological eradication, demonstrated by negative cultures of samples from all potentially infected mucosal sites at follow-up, is the sole determinant of efficacy.


Evaluation of new anti-infective drugs for the treatment of sexually transmitted chlamydial infections and related clinical syndromes. Infectious Diseases Society of America and the Food and Drug Administration. This guideline addresses clinical trials of new antimicrobial agents in the treatment of uncomplicated genital infections caused by Chlamydia trachomatis and of syndromes resembling chlamydial infections. The most common clinical manifestations of chlamydial infection are urethritis in men and mucopurulent cervicitis in women. However, many chlamydial infections are not associated with inflammatory symptoms or signs. Culture is the diagnostic standard for defining the presence of C. trachomatis, although nonculture tests may be used in screening patients for enrollment in clinical trials. Susceptibility testing for C. trachomatis is laborious and difficult to standardize; only a few clinical isolates need to be tested in vitro. Prospective, randomized, double-blind, active-control comparative studies are recommended. Eradication of C. trachomatis defines both microbiological success and overall cure for chlamydial infection, but clinical and nonmicrobiological laboratory criteria are paramount in assessing the therapeutic response in nonchlamydial urethritis or cervicitis.


Evaluation of new anti-infective drugs for the treatment of syphilis. Infectious Diseases Society of America and the Food and Drug Administration. Syphilis is caused by Treponema pallidum, a spirochetal bacterium pathogenic only for humans. The clinical course of disease is divided into three stages interspersed by periods of latency. Penicillin remains the treatment of choice for all stages of infection; tetracycline or erythromycin may be used as therapeutic alternatives in defined circumstances. Patients enrolled in clinical trials should be evaluated clinically, microscopically, and serologically for the presence of the spirochete. All participants, after undergoing counseling and giving informed consent, should be tested for infection with human immunodeficiency virus. Specific criteria exist for diagnosis of syphilis and response to therapy. It may be desirable to perform a small, uncontrolled, open trial of a new anti-infective drug for the collection of preliminary evidence of efficacy. A larger-scale, randomized, active-control comparative clinical trial is necessary to prove efficacy.


Evaluation of new anti-infective drugs for the treatment of selected infections of the skin and skin structure. Infectious Diseases Society of America and the Food and Drug Administration. No method for classifying infections of the skin and skin structure is uniformly accepted. Therefore, each protocol for the evaluation of new anti-infective drugs must include definitions of the skin and skin-structure infections to be treated. Clinical findings may suggest the etiology. Cultures should be performed by the best available technique. Because the yield of pathogens from sites of skin and skin-structure infection is often low, monitoring of the clinical response to therapy is paramount. Randomized, double-blind, active-control comparative studies are needed, and maintenance of blinding is recommended, even with a change in the route of administration of the study and/or control drugs. The period of follow-up should be defined for each type of infection.


Evaluation of new anti-infective drugs for the treatment of osteomyelitis in adults. Infectious Diseases Society of America and the Food and Drug Administration. Cases of osteomyelitis can be divided into four categories: acute hematogenous, vertebral, secondary to a contiguous focus of infection without vascular disease, and secondary to a contiguous focus of infection with vascular disease. Each category may be further divided into acute and chronic forms. Clinical symptoms persisting for > or = 10 days correlate roughly with the development of necrotic bone and chronic osteomyelitis. Patients enrolled in clinical trials should generally be > or = 12 years of age. Prior antimicrobial treatment does not exclude patients if the culture of a bone sample obtained at the time of enrollment yields pathogenic bacteria. Randomized, double-blind, active-control comparative studies are encouraged. Clinical outcome should be assessed during therapy and within 5-9 days, 4-6 weeks, and 11-13 months after completion of therapy. In the final assessment, clinical appraisal is paramount.


Evaluation of new anti-infective drugs for the treatment of acute hematogenous osteomyelitis in children. Infectious Diseases Society of America and the Food and Drug Administration. Most children with acute hematogenous osteomyelitis have no preceding illness. Their early symptoms are pain and fever. A bacterial etiology is established in approximately 75% of cases by needle aspiration of the affected site or blood culture. Clinical trials should be limited to cases of bacterial origin. The antimicrobial agents studied should be active against Staphylococcus aureus and streptococci. If children < 5 years of age are included, the drug should also be active against beta-lactamase-negative and -positive strains of Haemophilus influenzae. Randomized, prospective, double-blind comparative studies are preferable to open, evaluator-blinded trials. Clinical outcome is appraised by physical signs and symptoms. A successful microbiological outcome consists of presumptive eradication. The final assessment should be made 1 year after completion of therapy.


Evaluation of new anti-infective drugs for the treatment of infectious arthritis in adults. Infectious Diseases Society of America and the Food and Drug Administration. This guideline describes clinical trials of new anti-infective drugs for the treatment of septic arthritis due to bacteria other than Neisseria gonorrhoeae in adults. Septic arthritis is associated with fever and with physical findings at the affected joint. Diagnosis is established by culture of synovial fluid. Treatment includes the administration of antimicrobial drugs and drainage of the joint by needle aspiration or surgery. Multicenter, randomized comparative clinical trials that are single-, double-, or evaluator-blinded should be performed. However, an open trial of a new antimicrobial agent with historical controls is acceptable. Patients should receive treatment for at least 2-3 weeks. After 5 days of antimicrobial therapy, synovial fluid should be sterile and clinical signs and symptoms should have diminished. Patients should be followed for 2-4 weeks after completion of therapy.


Evaluation of new anti-infective drugs for the treatment of acute suppurative arthritis in children. Infectious Diseases Society of America and the Food and Drug Administration. Diagnostic criteria for bacterial suppurative arthritis include the demonstration of an inflammatory exudate by aspiration of synovial fluid and the isolation of bacteria from cultures of synovial fluid and/or blood. Clinical manifestations include joint effusion, swelling, tenderness, and pain, with or without redness of the overlying skin. Management consists of antimicrobial therapy, measures designed to relieve symptoms, surgical drainage of infected fluid, and physical therapy. Studies of new anti-infective therapy should be limited to cases of bacterial origin. Prospective, randomized, double-blind, or evaluator-blinded, active-control comparative clinical trials should be performed. Clinical response is characterized as success (cure), failure, or indeterminate outcome. The most common successful microbiological outcome is presumptive eradication. Follow-up should continue for 1 year before the final assessment.


Evaluation of new anti-infective drugs for the treatment of infections of prosthetic hip joints. Infectious Diseases Society of America and the Food and Drug Administration. Infections of a prosthetic hip are of three types: acute contiguous, chronic contiguous, and hematogenous. Acute contiguous infections result from contamination of the operative field at the time of surgery; clinical manifestations of infection become apparent within 6 months. Chronic contiguous infections are diagnosed 6-24 months postoperatively and are believed to be caused by intraoperative contamination. Hematogenous seeding of prosthetic joints accounts for infections that develop > or = 2 years after surgery. Fever and pain or dysfunction of the joint may be the only signs or symptoms of prosthetic hip joint infection. Definitive diagnosis is established by culture of a needle aspirate from the joint space or by intraoperative culture. Prospective, randomized, double-blind or evaluator-blinded, active-control comparative studies are preferable to open trials. Success rates 10-14 weeks after completion of a 4- to 6-week course of antimicrobial therapy should be > or = 90%.


Evaluation of new anti-infective drugs for the treatment of acute bacterial meningitis. Infectious Diseases Society of America and the Food and Drug Administration. Predisposing conditions for acute bacterial meningitis include prematurity, young age, management in an intensive care setting, low socioeconomic background, and crowded living conditions. Clinical findings vary with age and may be nonspecific (altered feeding behavior) or specific (Kernig and Brudzinski signs). Examination and culture of cerebrospinal fluid (CSF) are essential for diagnosis. Antigen identification in CSF, serum, or urine by latex agglutination or other techniques can be useful in the identification of the pathogen. Randomized, controlled studies with a single-, double-, or evaluator-blinded design are encouraged. Among neonates, infants, and children, CSF should be examined again 24-36 hours after initiation of therapy. Outcomes should be judged by both clinical and microbiological criteria. Assessment of microbiological outcome is paramount.


Evaluation of new anti-infective drugs for the treatment of cryptococcal meningitis. Infectious Diseases Society of America and the Food and Drug Administration. Cryptococcus neoformans may infect persons with intact or compromised host defenses. Clinical manifestations generally correlate directly with the degree of immunosuppression. Treatment is prescribed on the basis of the severity of disease and the degree of immunosuppression. Cryptococcal meningitis is the most common clinical form of cryptococcal infection and the most amenable to study in clinical trials. The current standard of care for cryptococcal meningitis is therapy with amphotericin B. Despite clinical improvement and microbiological suppression of the organism at the completion of therapy, the microbiological outcome will be identified as presumptive persistence if treatment is continued. Patients should be observed for 1 year after completion of therapy before a final assessment is made.


Evaluation of new anti-infective drugs for the treatment of viral encephalitis. Infectious Diseases Society of America and the Food and Drug Administration. Viral encephalitis may develop subsequent to viremia, via neuronal spread, or by arthropod vector. Diagnosis often requires invasive studies such as lumbar puncture and brain biopsy. This guideline addresses herpes simplex, rabies, and arbovirus infections of the central nervous system. Clinical trials should be designed according to the availability of approved therapeutic agents. Study designs with an active control (herpesvirus), a placebo control (arbovirus), or no control (rabies virus) are recommended. Outcome should be assessed 4-6 weeks, 4-6 months, and 11-13 months after the completion of therapy. For newborns with encephalitis, outcome should be assessed yearly through the age of 5 years. Assessment of clinical outcome is paramount.


Evaluation of new anti-infective drugs for the treatment of toxoplasma encephalitis. Infectious Diseases Society of America and the Food and Drug Administration. Toxoplasma gondii is a protozoan that exists in three forms, all of which are potentially infectious for humans. After acute infection, cysts persist in the central nervous system and extraneural tissue. Human hosts with compromised immunity, particularly those with the acquired immunodeficiency syndrome, are vulnerable to reactivation and dissemination. The most common clinical expression of toxoplasma infection is encephalitis. The diagnosis is established by clinical presentation, computed tomography and/or magnetic resonance imaging, and detection of antibodies to T. gondii in serum of patients positive for human immunodeficiency virus. Brain biopsy may be performed. Protocols may be developed for the evaluation of new regimens for the treatment of acute encephalitis, the suppression of disease after treatment, or the prevention of reactivation before the onset of clinical disease. Assessment of clinical outcome is paramount.


Evaluation of new anti-infective drugs for the treatment of febrile episodes in neutropenic patients. Infectious Diseases Society of America and the Food and Drug Administration. The use of empirical antimicrobial therapy has significantly reduced the morbidity and mortality associated with untreated infections in febrile neutropenic patients. This guideline describes clinical trials of the safety and efficacy of new antimicrobial drugs in this population of patients. Fever and neutropenia should be precisely defined in each protocol. Patients should be randomized to treatment with a new or active-control drug regimen, stratified on the basis of type of cancer and age, and treated until resolution--as defined in the protocol--is attained. Outcome should be assessed both for cases with a defined microbial etiology and for those without. Final microbiological outcome is important for cases with identified pathogens, but clinical outcome is paramount.


Evaluation of new anti-infective drugs for the treatment of urinary tract infection. Infectious Diseases Society of America and the Food and Drug Administration. The term urinary tract infection (UTI) encompasses a broad range of clinical entities that share one characteristic: a positive urine culture. Clinical manifestations and responses to therapy are diverse even when comparable numbers of a particular bacterial species are identified on urine culture. These guidelines include discussion of acute uncomplicated cystitis, acute uncomplicated pyelonephritis, and complicated infections of the urinary tract. It is proposed that the finding of > or = 10(3) cfu/mL of urine defines significant bacteriuria in acute uncomplicated cystitis, > or = 10(4) cfu/mL in acute uncomplicated pyelonephritis and UTI in men, and > or = 10(5) cfu/mL in complicated UTI. The preferred clinical study design is prospective, randomized, and controlled with an active agent. Ideally, treatment with antimicrobial agents should eradicate the infecting organism, bring about the resolution of clinical signs and symptoms, have few adverse effects, and prevent reinfection.


Evaluation of new anti-infective drugs for the treatment of acute infectious diarrhea. Infectious Diseases Society of America and the Food and Drug Administration. This guideline includes diarrhea causing infantile mortality in which a bacterial pathogen is recovered and for which oral rehydration therapy is an important component of care as well as traveler's diarrhea (with or without recovery of a pathogen). Diarrhea is defined as the passage of three or more unformed stools per day plus--in all patients except infants--one or more signs or symptoms of enteric infection. The preferred study design is prospective and randomized, with an active concurrent control and (when possible) blinding. Placebo-controlled trials may be performed if the severity of disease is judged by the investigator to be mild or moderate. It is desirable that both clinical and microbiological outcome be determined. Microbiological eradication is paramount.


Evaluation of new anti-infective drugs for the treatment of typhoid fever. Infectious Diseases Society of America and the Food and Drug Administration. Typhoid fever is an acute febrile illness caused by Salmonella typhi. The evidence of blood-borne infection required for study entry includes clinical signs and symptoms plus confirmation of the presence of S. typhi in blood or other tissues or body fluids. The preferred study design is prospective and randomized with an active concurrent control. It is preferred that the investigator or an evaluator be blinded to therapy. In general, treatment should be administered for 2 weeks until it is demonstrated that a shorter course is as efficacious and as safe. Follow-up cultures of specimens from sites originally shown to be infected with S. typhi should be performed unless the diagnostic procedure places the patient at unnecessary risk.


Evaluation of new anti-infective drugs for the treatment of cholera. Infectious Diseases Society of America and the Food and Drug Administration. Cholera is an acute gastrointestinal infection caused by Vibrio cholerae. It is characterized by watery diarrhea that may lead to massive fluid loss, which in turn may result in hypotension, shock, and death within hours. Key to the treatment of cholera is fluid replacement. Anti-infective therapy decreases the severity and duration of diarrhea and the duration of shedding of V. cholerae. Enrolled patients should have diarrhea that is moderate to severe and a culture that ultimately yields V. cholerae. A prospective, randomized, active-controlled clinical trial is preferred. Studies should be double-blinded or evaluator-blinded. The rapidity with which the organism is eliminated from stool may be assessed. Both clinical and microbiological outcome should be determined. Assessment of microbiological eradication is paramount, since fluid replacement may suffice for treatment of signs and symptoms.


Evaluation of new anti-infective drugs for the treatment of diarrhea caused by Giardia lamblia. Infectious Diseases Society of America and the Food and Drug Administration. Giardia lamblia is a flagellate protozoan that produces symptoms by infecting the small bowel and biliary tract in the trophozoite form. Diagnosis is currently established by microscopic visualization of the organism in appropriate intestinal contents (stool, small-bowel contents, or biopsy specimen). Adult patients with diarrhea and one or more enteric symptoms may be enrolled in clinical trials of new drugs for the treatment of giardial disease. A randomized, double-blind, active-concurrent-control design is recommended. Post hoc stratification by age, immune status, chronicity of disease, and ease of establishing diagnosis (organism load) may be performed. Microbiological assessment 48 hours to 7 days after the completion of therapy is paramount for determining final outcome.


Evaluation of new anti-infective drugs for the treatment of diarrhea caused by Cryptosporidium. Infectious Diseases Society of America and the Food and Drug Administration. Cryptosporidium is a coccidian protozoan that produces symptoms by infesting the small bowel. The illness is characterized by watery stools, anorexia, weight loss, and abdominal pain. Diagnosis is made by visualization of the organisms on microscopic examination of stool. There currently is no approved therapy for this infection. A randomized, double-blind, placebo-controlled study design is recommended. Stratification of patients by age and immune status should be considered. Two stool samples obtained 48 hours to 7 days after completion of therapy should be negative for Cryptosporidium oocysts. Assessment of microbiological outcome is paramount.


Evaluation of new anti-infective drugs for the treatment of diarrhea caused by Entamoeba histolytica. Infectious Diseases Society of America and the Food and Drug Administration. Entamoeba histolytica causes colonic infection that ranges from asymptomatic carriage to invasive disease with infection of extraintestinal organs, particularly the liver. The disease occurs in both sporadic and epidemic forms. Diagnosis requires visualization of trophozoites or cysts by microscopic examination of stool, colonic scrapings, or biopsy specimens. Patients with either asymptomatic or symptomatic disease may be eligible for clinical trials. A prospective, randomized, double-blind, placebo-controlled study design is recommended for asymptomatic carriers and an active-concurrent-control study design for symptomatic patients. Final outcome should be assessed 48 hours to 7 days after completion of therapy. Assessment of microbiological outcome is paramount.


Evaluation of new anti-infective drugs for the treatment of chronic carriage of Salmonella. Infectious Diseases Society of America and the Food and Drug Administration. The chronic carriage of salmonellae is defined as the shedding of a Salmonella species for > or = 1 year, as documented by an initial positive culture of a stool sample obtained at least 1 month after resolution of the acute illness and repeated positive cultures for at least 1 year. Clinical trials of investigational anti-infective drugs for the treatment of the salmonella carrier state may be conducted with a placebo control or an active concurrent control. A crossover design also may be employed for establishing efficacy. Patients should generally receive therapy for at least 6 weeks. Outcome will be assessed only by microbiological criteria. Determination of the interval required for the suppression of salmonellae and follow-up for 6 months after completion of therapy are recommended.


Evaluation of new anti-infective drugs for the treatment of antibiotic-associated colitis. Infectious Diseases Society of America and the Food and Drug Administration. Colitis due to Clostridium difficile is diagnosed in 10%-15% of hospitalized patients who develop diarrhea after treatment with antimicrobial drugs. Diagnosis is based on the concurrence of diarrhea, one or more signs or symptoms of enteric intoxication, and stool from which toxigenic C. difficile is isolated or from which its toxins are identified. Clinical trials evaluating therapy may be placebo controlled (for mild disease) or concurrently controlled with an active drug. A randomized, double-blind study design is preferred. Outcome should be assessed by monitoring of the degree of inflammation of the bowel mucosa, the intensity and severity of diarrhea, the duration of illness, changes in stool form, and the eradication of C. difficile or its toxins from stool. Because C. difficile can be a component of the normal gastrointestinal flora, assessment of clinical outcome is paramount.


Evaluation of new anti-infective drugs for the treatment of gastritis and peptic ulcer disease associated with infection by Helicobacter pylori. Infectious Diseases Society of America and the Food and Drug Administration. Helicobacter pylori is a gram-negative, microaerophilic, spiral bacillus. Infection by this organism is currently believed to be the major cause of type B gastritis. Inflammation and infection may persist for years in the absence of therapeutic intervention. There is currently no approved antimicrobial therapy for gastritis. Clinical investigations have shown that combination regimens including bismuth salts and antimicrobial drugs result in the relief of symptoms, the resolution of histologic evidence of gastritis, the eradication of H. pylori, high rates of ulcer healing, and lower rates of ulcer relapse than have been found with other therapies (antacids and H2 antagonists). A randomized, double-blind, placebo-controlled study design is recommended for evaluation of new therapies. Study participants should have their progress monitored by endoscopy performed at enrollment, at completion of therapy, and 3 months thereafter. Assessment of microbiological outcome is paramount for final evaluation of the patient.


Evaluation of new antifungal drugs for the treatment of systemic fungal infections. Infectious Diseases Society of America and the Food and Drug Administration. These guidelines are applicable to all fungal pathogens that produce systemic infections in humans. Specific examples are provided whenever they might clarify special issues. Systemic fungal infections usually are divided into two broad categories: endemic systemic fungal diseases, which occur classically in healthy hosts, and opportunistic fungal diseases, which occur almost exclusively in patients with impaired host defenses. Both the increasing frequency of disseminated histoplasmosis and coccidioidomycosis in patients with AIDS and the occurrence of candidemia due to vascular-line infections have begun to blur this distinction. The fungi included in these guidelines are Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Candida species, Cryptococcus neoformans, Aspergillus species, and Sporothrix schenckii. Diagnosis of infections caused by these fungi should be based on culture of infected body fluids or tissues whenever possible. Cryptococcal and coccidioidal meningitis are exceptions. Amphotericin B remains the standard comparative agent for most new agents. Further studies of the efficacy of new oral agents used alone or after a hospital course of amphotericin B are needed. The agents currently available are usually inadequate for eradication of fungal infections in patients with AIDS, who may need prolonged treatment. Final assessment for these patients may need to be classified as clinical cure with presumed microbiologic persistence.


Evaluation of new anti-infective drugs for the treatment and prevention of tuberculosis. Infectious Diseases Society of America and the Food and Drug Administration. This guideline addresses the evaluation of new antimycobacterial drugs in the treatment and prevention (secondary prophylaxis) of infection by M. tuberculosis. Patients may be enrolled in clinical trials on the basis of clinical and/or microbiological criteria. A therapeutic regimen will likely include a combination of drugs; a randomized, active-control, comparative clinical trial is recommended. If appropriate samples can be obtained for culture during follow-up without placing the patient at unwarranted risk, the assessment of microbiological outcome is paramount. Prophylaxis will probably require a single drug, and a similar study design is preferred.


Evaluation of new anti-infective drugs for the treatment and prevention of infections caused by the Mycobacterium avium complex. Infectious Diseases Society of America and the Food and Drug Administration. The bacteria of the Mycobacterium avium complex are ubiquitous; thus it is often difficult to distinguish environmental contamination from colonization or infection. Patients with either pulmonary or disseminated infection may be enrolled in clinical trials. Disseminated disease occurs mostly in patients infected with the human immunodeficiency virus. In general, a randomized, active-control, double-blinded clinical trial is preferred; there should at least be a blinded evaluator. With regard to immunosuppressed populations, new antimycobacterial drugs need to be evaluated not only for the treatment but also for the prevention of disease. For trials of prophylaxis a placebo-controlled design is ethical until a drug is proven effective; then the use of an active-control regimen is appropriate. Since no regimen has been approved by the U.S. Food and Drug Administration for treatment or prevention of disease caused by the M. avium complex, demonstration of the superiority of the study regimen to the control regimen should be the objective of the clinical trial.


Evaluation of new anti-infective drugs for the treatment of disease caused by Mycobacterium kansasii and other mycobacteria. Infectious Diseases Society of America and the Food and Drug Administration. Mycobacterium kansasii is a photochromogenic nontuberculous mycobacterium that usually causes infections of the respiratory tract in humans. Although spontaneous resolution of infection has been reported, most patients require antimycobacterial therapy. A three- or four-drug combination--isoniazid, rifampin, and ethambutol and/or streptomycin--usually is prescribed. For evaluation of a new drug, a randomized, double-blind or evaluator-blinded, active-control comparative study design is recommended. Treatment should continue for 18-24 months, and follow-up evaluations should be conducted every 6 months for 3 years. Microbiological outcome is paramount.


Evaluation of new anti-infective drugs for surgical prophylaxis. Infectious Diseases Society of America and the Food and Drug Administration. It has been established by substantial research that antimicrobial prophylaxis for various surgical procedures can reduce the risk of postoperative morbidity and mortality. When the incidence of infectious complications is high, the reduction with prophylaxis is most dramatic. However, even for many "clean" procedures (vascular procedures, total joint replacement), the small reduction in potentially calamitous complications justifies the use of prophylaxis. Many issues of detail remain unanswered: timing and duration of administration of antimicrobial drug; type of drug; use of topical anti-infective agents as ancillary measures; and choices for high-risk individuals and others ordinarily excluded from clinical trials. An approach to the conduct of clinical trials of anti-infective drugs for surgical prophylaxis is provided. Both general guidelines and specific recommendations for total hip replacement, colorectal operations, appendectomy, and transurethral resection of the prostate are included.


Evaluation of new anti-infective drugs for the treatment of intraabdominal infections. Infectious Diseases Society of America and the Food and Drug Administration. These guidelines deal with the evaluation of anti-infective drugs for the treatment of intraabdominal infections. The clinical entities consist of infections arising from any part of the gastrointestinal tract, from the distal esophagus to the colon. These include surgical infections of the bowel, biliary tree, liver, spleen, and pancreas. Virtually all intraabdominal infections are due to multiple microorganisms resident in the gastrointestinal tract; these include aerobes and facultative and obligate anaerobes. Infections are classified as complicated (requiring an operative procedure), uncomplicated (managed medically), and postoperative wound (the operative procedure should be curative, but anti-infective drugs are used to prevent further infection at the site). Clinical criteria are paramount for entry into a study and for evaluation of efficacy. For complicated infections an adequate operation is an important determinant of outcome and needs assessment. Cultures of purulent intraabdominal fluid or abscess material are the only valid microbiologic indicators of infection. The acute physiology and chronic health evaluation score is useful in defining the severity of acute illness. The control regimen should consist of effective, established drugs and surgical procedures for the condition. Duration of therapy for complicated infections is usually 5-14 days; for uncomplicated infections, 3-7 days; and for postoperative wound infection, 2-5 days. Periodic assessment of safety and efficacy must be conducted during therapy. The outcome at final assessment is cure, failure, or indeterminate.


General guidelines for clinical bacteriology. Infectious Diseases Society of America and the Food and Drug Administration. This guideline summarizes recommendations for (1) developing cogent procedures for diagnosis and antimicrobial susceptibility testing; (2) developing quality-control parameters for the microbiological components of clinical trials; (3) continually updating U.S. Food and Drug Administration (FDA) guidelines; (4) reviewing microbiological recommendations from other groups, such as Microbiology Subcommittees of the National Committee for Clinical Laboratory Standards; and (5) improving the microbiological aspects of FDA package inserts for antimicrobial drugs. Sensitive and specific methods for isolation and identification of pathogens are essential to the proper conduct of clinical trials. Susceptibility tests should be performed in an accurate and reproducible fashion. Verification of results in a reference laboratory is encouraged to monitor quality control.


Evaluation of new anti-infective drugs for the treatment of acute pelvic infections in hospitalized women. Infectious Diseases Society of America and the Food and Drug Administration. This set of guidelines deals with evaluation of anti-infective drugs for treatment of acute pelvic infections in hospitalized women. The clinical entities include infectious complications of cesarean section; elective hysterectomy; and septic, incomplete abortion. Conditions including endomyometritis, cuff cellulitis, pelvic cellulitis, parametritis, phlegmon, and pelvic abscesses may arise due to a variety of bacterial species, both aerobic and anaerobic, that comprise the endogenous flora of the lower reproductive tract. Anaerobic bacteria have assumed particular importance, and therapy should be directed against such organisms. The roles of enterococci, chlamydiae, and mycoplasmas remain uncertain. Culture samples must be obtained under conditions assuring minimal vaginal contamination. Before a new drug may be used for treatment of human pelvic infections, considerable information is necessary about its antimicrobial spectrum as well as its safety and efficacy. Placebo-controlled trials are considered unethical. Historical controls may be used, but concurrent active control comparative trials are preferred. Parenteral administration is recommended for at least the initial 4 days of therapy, but orally administered drugs may be evaluated for completion of longer courses. The expected cure rate is approximately 90%. Uncomplicated infections should be treated for at least 4 days; more complicated infections may require prolonged therapy. Although clinical cure is paramount, microbiologic response must also be taken into account. In the final assessment, outcome will be classified as cure, failure, or indeterminate.


General guidelines for the clinical evaluation of anti-infective drug products. Infectious Diseases Society of America and the Food and Drug Administration. This document provides new general guidelines for the design and execution of studies evaluating anti-infective drugs for the prevention or treatment of infectious diseases. The first step in evaluation is the determination of in vitro microbial susceptibility. Next, studies are conducted in animals. Several animal models provide information useful in the prediction of appropriate dosing and activity in humans. If the results of these studies are favorable, staged clinical trials are then conducted. These guidelines reflect changes in the practice of medicine, dealing with topics such as the switch from parenteral to oral drug administration during a course of therapy, treatment in settings other than acute-care hospitals, and the use of alternative comparison drugs for the study of indications or dosing schedules not covered by the product label. Because further changes in practice are anticipated, the present guidelines will need to be updated and revised periodically.


Evaluation of new anti-infective drugs for the treatment of acute pelvic inflammatory disease. Infectious Diseases Society of America and the Food and Drug Administration. Pelvic inflammatory disease (PID) is a syndrome unrelated to pregnancy or surgery and characterized by lower abdominal pain and tenderness, cervical motion tenderness, and adnexal tenderness. Fever, leukocytosis, and the results of laboratory tests are used to support the diagnosis. Participants in clinical trials should be stratified into two groups: those with and those without tubo-ovarian abscess--i.e., those with complicated and those with uncomplicated PID. Diagnostic studies and treatment should be directed at four major groups of pathogens: Neisseria gonorrhoeae, Chlamydia trachomatis, anaerobic bacteria, and facultative bacteria such as Escherichia coli. Women requiring hospitalization should generally be treated as inpatients for at least 4-7 days; outpatient therapy should then be instituted to complete a 14- to 21-day course. Clinical and laboratory evaluations should be conducted daily during hospitalization and both 2-4 days and 2-4 weeks after the completion of therapy.


Evaluation of new anti-infective drugs for the treatment of respiratory tract infections. Infectious Diseases Society of America and the Food and Drug Administration. These guidelines deal with the evaluation of anti-infective drugs for the treatment of respiratory tract infections. Five clinical entities are described: streptococcal pharyngitis and tonsillitis, otitis media, sinusitis, bronchitis, and pneumonia. A wide variety of microorganisms are potentially pathogenetic in these diseases; these guidelines focus on the bacterial infections. Inclusion of a patient in a trial of a new drug is based on the clinical entity, with the requirement that a reasonable attempt will be made to establish a specific microbial etiology. Microbiologic evaluation of efficacy requires isolation of the pathogen and testing for in vitro susceptibility. Alternatively, surrogate markers may be used to identify the etiologic agent. The efficacy of new drugs is evaluated with reference to anticipated response rates. Establishment of the microbial etiology of respiratory tract infections is hampered by the presence of "normal flora" of the nose, mouth, and pharynx, which may include asymptomatic carriage of potential pathogens. This issue is addressed for each category of infection described. For example, it is suggested that for initial phase 2 trials of acute otitis media and acute sinusitis tympanocentesis or direct sinus puncture be used to collect exudate for culture. Acute exacerbations of chronic bronchitis also present difficulties in the establishment of microbial etiology. These guidelines suggest that clinical trials employ an active control drug but leave open the possibility of a placebo-controlled trial. For pneumonia, the guidelines suggest the identification and enrollment of patients by the clinical type of pneumonia, e.g., atypical pneumonia or acute bacterial pneumonia, rather than by etiologic organism or according to whether it was community or hospital acquired. For each respiratory infection, the clinical response is judged as cure, failure, or indeterminate. Clinical improvement is not acceptable unless quantitative response measures can be applied.


Evaluation of new anti-infective drugs for the treatment of infective endocarditis. Infectious Diseases Society of America and the Food and Drug Administration. These guidelines describe the design and implementation of clinical trials to assess the safety and efficacy of anti-infective drugs for the treatment of infective endocarditis. Identification and enrollment of patients in clinical trials is based on a modification of traditional criteria. To accrue a sufficient number of patients, only those with streptococcal or staphylococcal endocarditis should be included in studies. Results of treatment with approved drugs allow for projection of expected bacteriologic cure rates and survival rates. Prospective randomized, double-blind studies are recommended. These guidelines are based on the premise that future protocols may include shorter courses of therapy, combinations of drugs, or progression from parenteral to oral therapy. Clinical response is judged as cure, failure, or indeterminate; there is no "improved" category. Microbiologic response is categorized as eradication, persistence, or relapse. When a patient has shown no clinical evidence of active disease for a protracted period, there may be no need to perform a posttreatment blood culture; for such patients, the microbiologic response is termed presumptive eradication. Several months of follow-up may be necessary to detect late relapses.


Special issues in clinical trials of new anti-infective drugs for the treatment of sexually transmitted diseases. Infectious Diseases Society of America and the Food and Drug Administration. Several special issues arise in relation to clinical trials of therapy for sexually transmitted diseases. These issues include the desirability of including adolescents and both pregnant and nonpregnant women in the trial, the use of unapproved control regimens, problems with antimicrobial susceptibility testing due to inadequate methodology and the need for prompt treatment, the need to assess agents for treatment of syndromes of unknown microbial etiology, toxicity considerations related to the use of single-dose regimens, management of the sexual partners of the participants in the trial, analysis of data despite the high frequency of minor protocol violations, sexual reexposure to infection during the trial, and the potential for loss, alteration, or falsification of data because of the relative simplicity of the usual protocol design and the diagnostic reliance on specimens that are routinely discarded.


Evaluation of new anti-infective drugs for the treatment of genital infections due to herpes simplex virus. Infectious Diseases Society of America and the Food and Drug Administration. This guideline addresses clinical trials of new therapies for genital infections due to herpes simplex virus (HSV). Of the two types of virus, HSV-2 is the more common pathogen. Both HSV-1 and HSV-2 become latent in sacral nerve root ganglia and intermittently reactivate. Patients who have frequent recurrences (more than four per year) may be candidates for long-term suppressive therapy. In both first-episode and recurrent genital HSV infections, lesions should be cultured for HSV. Testing for human immunodeficiency virus is encouraged but not required. Serum antibodies to HSV-1 and HSV-2 should be quantitated at enrollment and 3-5 weeks thereafter. Randomized, double-blind, active-control comparative studies are generally recommended. Placebo-controlled trials may be appropriate for recurrent genital herpes or for suppression of recurrences. Final evaluation should generally take place 10-15 days after the completion of therapy.


Solidifying liquid with novel initiation system for detachable balloon catheters. In endovascular surgery, to permanently hinder blood inflow into an aneurysm for example, a catheter is percutaneously introduced into an artery and the balloon is positioned and inflated by a solidifying liquid and then released into the diseased part. A novel solidifying liquid for the inflated balloon curable under the continuous feeding of oxygen through the wall of the balloon is described here. Glucose oxidase which oxidizes glucose producing hydrogen peroxide was used for polymerization of the solidifying liquid. This solidifying liquid cured even in the presence of oxygen and water. It was radiopaque when mixed with the iodinated X-ray contrast chemical iopamidol. In addition, the solidified liquid had appropriate mechanical properties. The endovascular surgery could be carried out safely using it.


Influence of substratum wettability on the strength of adhesion of human fibroblasts. To determine the strength of adhesion and the detachment mechanisms of fibroblasts from substrata with different wettability, the behaviour of adhered cells was studied in a parallel-plate flow chamber during exposure to shear. Adhered cells were observed in situ, i.e. in the flow chamber, by phase-contrast microscope and images were analysed semiautomatically. Detachment was found to be dependent both on shear stress and time, although a critical shear stress can be found below which no detachment occurs. On all substrata, cells round up before detachment and are approximately spherical immediately before detachment. The strength of adhesion calculated ranged from 0.6-3.5 x 10(-10) N per cell on FEP-Teflon (the least wettable material included) to 9.4 x 10(-9) N per cell for glass (the most wettable). Ease of detachment seemed to decrease with increasing wettability. However, cells reacted more strongly with tissue culture polystyrene (TCPS) than expected on the basis of its wettability, probably due to surface chemistry.