Dynamics of pituitary down-regulation and ovarian response to controlled stimulation in in vitro fertilization cycles.

The effect of the daily GnRH agonist administration on LH levels and ovarian response after down-regulation was investigated in 2 groups of women who received recombinant FSH versus hMG. The results showed that the pituitary gland remained responsive to the daily buserelin by producing pulses of LH, despite down-regulation in both groups; however, the patients whose condition is profoundly down-regulated may need exogenous LH to improve their response.

Antimüllerian hormone and pituitary gland activity after prolonged down-regulation with goserelin acetate.

The size of the pool of growing follicles was normal after prolonged down-regulation, as indicated by normal AMH levels 4 and 8 weeks after goserelin administration. However, there was a profound down-regulation of LH levels; therefore we suggest administration of exogenous LH to proceed to IVF or alternatively stimulation of endogenous LH secretion with daily administration of GnRH agonist. These need to be assessed prospectively.

Agonist "flare-up" versus antagonist in the management of poor responders undergoing in vitro fertilization treatment.

OBJECTIVE: To compare the agonist flare-up and antagonist protocols in the management of poor responders to the standard long down-regulation protocol. DESIGN: Retrospective comparative study. SETTING: Assisted conception center. PATIENT(S): One hundred thirty-four patients undergoing IVF/ intracytoplasmic sperm injection (ICSI) treatment, who responded poorly to the standard long down-regulation protocol in their first treatment cycle. In the second cycle, 77 received short flare-up agonist and 57 received antagonist protocol. We analyzed the outcome of the second cycle. INTERVENTION(S): Peak serum E(2) was assayed on the day of hCG administration. MAIN OUTCOME MEASURE(S): Cycle cancellation rate due to poor ovarian response. RESULT(S): There was no cycle cancellation in the flare-up protocol and 7% cancellation rate in the antagonist protocol due to lack of response. A significantly higher number of patients had embryo transfer in the flare-up protocol. There was no difference in pregnancy rate (PR) between the two groups. CONCLUSION(S): Both the flare-up and the antagonist protocols significantly improved the ovarian response of known poor responders. However, a significantly higher cycle cancellation rate and less patients having embryo transfer in the antagonist group tips the balance in favor of the flare-up protocol.

Expression of the mRNAs and Proteins for the Na(+)/H(+) exchangers and their regulatory factors in baboon and human placental syncytiotrophoblast.

In polarized epithelial cells of several organ systems, e.g. the kidney, a family of Na(+)/H(+) exchangers (e.g. Na(+)/H(+) exchanger-1 and -3) and their regulatory proteins, Na(+)/H(+) exchanger regulatory factor and Na(+)/H(+) exchanger-3 kinase A regulatory protein play a major role in regulating Na(+)/H(+) exchange integral to cellular homeostasis. Because the primate placenta regulates exchange of Na(+) and H(+) between the mother and fetus critical to fetal-placental homeostasis, the current study determined whether Na(+)/H(+) exchanger-1 and -3 were compartmentalized and associated with expression of Na(+)/H(+) exchanger regulatory factor and Na(+)/H(+) exchanger-3 kinase A regulatory protein in baboon and human syncytiotrophoblast. Using RT-PCR, single 413-bp Na(+)/H(+) exchanger-1 and 190-bp Na(+)/H(+) exchanger-3 products were expressed by baboon and human syncytiotrophoblasts. The 104-kDa Na(+)/H(+) exchanger-1 protein was detected by Western blot in microvillus membranes and to a much lesser extent in the basal membranes of the baboon and human syncytiotrophoblasts. In contrast, the 85-kDa Na(+)/H(+) exchanger-3 protein was detected primarily in membranes contiguous with the basal membranes of the syncytiotrophoblast of both species. Differential localization of Na(+)/H(+) exchanger-1 and -3 was confirmed by immunocytochemistry. The Na(+)/H(+) exchanger-3 regulatory protein, Na(+)/H(+) exchanger-3 kinase A regulatory protein, resided almost exclusively in the basal membranes, whereas Na(+)/H(+) exchanger regulatory factor was localized primarily to the microvillus membranes in the baboon and human syncytiotrophoblast. Collectively, these results are the first to show that the baboon and human term placental syncytiotrophoblast expressed the mRNAs and proteins for Na(+)/H(+) exchanger-1 and -3 and their regulatory factors and that Na(+)/H(+) exchanger-1 and Na(+)/H(+) exchanger regulatory factor resided primarily in the microvillus membranes, whereas Na(+)/H(+) exchanger-3 and Na(+)/H(+) exchanger-3 kinase A regulatory protein were localized to membranes contiguous with the basal membranes and to the basal membranes, respectively. We conclude that a complete Na(+)/H(+) exchange system is present in the baboon and human term placental syncytiotrophoblast and suggest that the primate placenta exhibits polarity with respect to the capacity for regulation of Na(+)/H(+) exchange between the placenta and the maternal and fetal circulations.

Identification and developmental expression of the estrogen receptor alpha and beta in the baboon fetal adrenal gland.

We have previously shown that estrogen regulates the development and function of the fetal and definitive/transitional zones of the primate fetal adrenal gland. Thus, during baboon pregnancy estrogen acts directly on the fetal zone to suppress ACTH-stimulated dehydroepiandrosterone (DHA) formation, potentially to modulate C19-steroid production and consequently placental estrogen synthesis. It is proposed that this action of estrogen is mediated by the estrogen receptor. Therefore, in the present study a developmental approach was used to determine whether the messenger RNA (mRNA) and protein for the estrogen receptor were expressed in the fetal and definitive/transitional zones ofthe baboon fetal adrenal gland at mid (day 100) and late (day 170) gestation (term = 184 days). Estrogen receptor alpha mRNA levels, determined by competitive RT-PCR, were approximately 7-fold greater (P < 0.02) in the fetal adrenal of late (187.8+/-40.3 attomoles/microg RNA) compared with mid (27.4+/-5.4 attomoles/microg RNA) gestation. Moreover, estrogen receptor alpha mRNA expression, determined by quantitative in situ hybridization, was approximately 2.5-fold greater (P < 0.05) in the definitive/transitional zones (21.6+/-0.5 silver grains/0.025 mm2) than in the fetal zone (8.3+/-1.5 grains/0.025 mm2) late in gestation. The mRNA for the beta-isoform of the estrogen receptor was also expressed in the baboon fetal adrenal cortex. There was a gradient of immunocytochemical staining for the estrogen receptor alpha and beta proteins, with extensive immunoreactivity for both isoforms in the definitive zone and lower staining in the transitional zone and the fetal zone. In summary, the results of the present study show that estrogen receptor alpha and beta were expressed in the fetal and definitive/transitional zones of the baboon fetal adrenal cortex at mid and late gestation. The presence of the estrogen receptor provides a mechanism for mediating the action of estrogen in modulating ACTH-dependent and cortical zone-specific development and function of the primate fetal adrenal gland.

Cloning of the 11beta-hydroxysteroid dehydrogenase (11beta-HSD)-2 gene in the baboon: effects of estradiol on promoter activity of 11beta-HSD-1 and -2 in placental JEG-3 cells.

In the baboon, estrogen regulated 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzed metabolism of cortisol and cortisone by the placenta is an important component in the sequence of events regulating the fetal pituitary-adrenocortical axis. The present study was designed to isolate and sequence the promoter region of the baboon 11beta-HSD-2 gene and to produce constructs of this gene and the 1.7 kb fragment of 5'-flanking region of baboon 11beta-HSD-1 isolated previously in order to determine whether the promoters of these two genes were activated in human placental JEG-3 cells and whether expression could be modulated by estradiol. The 11beta-HSD-2 genomic DNA was isolated from a baboon kidney genomic library using a human 11beta-HSD-2 cDNA as a probe. The sequence of a 1.2 kb fragment of the 5'-flanking region showed extensive homology with that published by others for human 11beta-HSD-2, particularly in exon 1 (>95%) and in the proximal promoter (>90%). Primer extension confirmed that the baboon 11beta-HSD-2 gene has multiple transcriptional start sites which are preceded by a GC box. To determine promoter activity of 11beta-HSD-2 and -1, the 5'-flanking regions of these genes were subcloned into luciferase reporter pGL3 vectors, transiently transfected into human placental JEG-3 cells, and then incubated for 16-18 h in the presence or absence of 10-8 M 17beta-estradiol or 17alpha-estradiol. To augment the low level of estrogen receptor (ER) in JEG cells, promoter activity studies were also performed in JEG cells co-transfected with an expression vector containing the human ER cDNA. The promoters of both 11beta-HSD-1 and -2 were activated following transient transfection into JEG-3 cells although basal activity of 11beta-HSD-2 (87+/-21 RLU/microg protein) always exceeded (P<0.05) that of 11beta-HSD-1 (37+/-7). In the absence of co-transfected ER, basal promoter activities of both 11beta-HSD genes were not altered by 17beta-estradiol. In contrast, in cells co-transfected with ER, 17beta-estradiol but not 17alpha-estradiol increased (P<0.05) basal promoter activities of 11beta-HSD-1 and -2 by 8.1+/-1.5 and 8.3+/-2. 0 fold, respectively. Collectively, these findings indicate that the promoter region of the baboon 11beta-HSD-2 gene is comparable to that in the human and that the 5'-flanking region of both the baboon 11beta-HSD-1 and -2 genes were active when transiently transfected into JEG-3 cells and that activation could be enhanced by estradiol in the presence of an estrogen receptor.

Decline in adrenocorticotropin receptor messenger ribonucleic acid expression in the baboon fetal adrenocortical zone in the second half of pregnancy.

We have previously shown a decrease in fetal zone-specific ACTH-stimulable dehydroepiandrosterone formation and an increase in definitive zone-specific cortisol biosynthesis in the baboon fetal adrenal gland in the second half of gestation. Therefore, the fetal and definitive zones seem to develop a divergence in functional capacity with advancing gestation. We have proposed, therefore, that there is a selective decrease in ACTH receptor expression and thus tropic responsivity to ACTH within the fetal zone in the second half of primate pregnancy. The present study examined this possibility and whether corresponding changes occurred in the developmental expression of major components required for steroidogenesis. ACTH receptor messenger RNA (mRNA) levels, determined by in situ hybridization, in the fetal zone of the baboon fetal adrenal were approximately 2-fold greater (P < 0.05) at mid (i.e. day 100) than at late (i.e. day 170) gestation and 3-fold greater (P < 0.01) in the definitive zone than in the fetal zone in late gestation (term = 184 days). Both ACTH receptor and low density lipoprotein receptor mRNA levels, determined by Northern blot in the whole fetal adrenal, also decreased (P < 0.001) by approximately 50%, whereas the mRNA levels for the definitive zone-specific delta5-3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) enzyme required for cortisol biosynthesis increased over 13-fold (P < 0.001) between mid and late gestation. In contrast, mRNA expression of the steroidogenic enzymes P-450 cholesterol side-chain cleavage and 17alpha-hydroxylase/17-20 lyase were unchanged throughout gestation. We conclude that the decrease in ACTH receptor mRNA expression and ACTH-stimulable dehydroepiandrosterone formation in the second half of gestation reflect a decline in functional capacity of the fetal zone, whereas the increase in 3beta-HSD mRNA expression and cortisol production results from the ACTH receptor-mediated development and enhanced functional capacity of the definitive zone.

Cloning and expression of the 11beta-hydroxysteroid dehydrogenase type 1 gene in the baboon.

In the baboon, the 11beta-hydroxysteroid dehydrogenase (11beta-HSD)-catalyzed metabolism of maternal cortisol and cortisone by the placenta is an important component in the sequence of events regulating the function of the fetal pituitary-adrenocortical axis. The present study was designed to isolate and sequence the promoter region of the baboon 11beta-HSD-1 gene. The 11beta-HSD-1 genomic DNA was isolated from a baboon kidney genomic library using a human 11beta-HSD-1 cDNA as a probe. The sequence of a 1.7 kb fragment of the 5'-flanking region showed extensive homology (> 95) to that published by others for human 11beta-HSD-1 particularly in exons I and II (> 95%) and in the proximal promoter ( > 98%). Using total RNA from adult baboon liver annealed with a 22 bp antisense primer located at the 3' end of Exon I, parallel genomic sequencing reactions with the same primer confirmed that the baboon 11beta-HSD-1 gene has two transcriptional start sites 93 nucleotides apart and that both start sites are preceded by a CAAT box but not a TATA box. RNase protection assays confirmed that both transcription start sites are utilized in liver and near term baboon placenta and that transcripts emanating from the downstream start site dominate in the placenta in contrast to the preferential utilization of the upstream start site in adult liver.

Hypothalamic corticotropin-releasing hormone expression in the baboon fetus at mid- and late gestation.

We have proposed that estrogen, via regulation of the placental 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) enzyme(s) catalyzing the oxidation of cortisol to its inactive metabolite cortisone, regulates the baboon fetal pituitary-adrenocortical axis and the onset of de novo production of cortisol by the fetus near term. In support of this hypothesis we have demonstrated that the increase in expression of the mRNA for the ACTH precursor proopiomelanocortin (POMC) in the fetal pituitary and in the specific activity of steroidogenic enzymes in the fetal adrenal normally observed at term were enhanced at midgestation by maternal estrogen administration. However, it is not known whether activation of the fetal pituitary reflects a concomitant increase in corticotropin-releasing hormone (CRH) mRNA expression and/or peptide production by the fetal hypothalamus. Therefore, an aim of the present study was to determine whether the increase in POMC mRNA in fetal baboons delivered at term, and at midgestation to mothers treated with estradiol, reflected an increase in hypothalamic CRH. Fetal hypothalami were obtained on Day 100 (n = 6) and Day 165 of gestation (term = Day 184) from untreated baboons (n = 5) and on Day 100 from baboons (n = 4) whose mother had been treated daily with 1.0 mg estradiol on Days 70 to 100. Hypothalamic CRH peptide concentrations were determined by RIA, and CRH mRNA expression was quantified by in situ hybridization in sections of the fetal hypothalamus through the paraventricular nucleus (PVN) using a 48-base synthetic oligodeoxynucleotide probe 3' end-labeled with [35S]dATP. The mean (+/- SE) maternal serum estradiol concentration in baboons treated with estradiol at midgestation (2.4 +/- 0.4 ng/ml) was greater (p < 0.05) than that in untreated baboons on Day 100 (1.0 +/- 0.2), but similar to that in late gestation (2.0 +/- 0.2). The mean steady-state concentration of CRH in the baboon fetal hypothalamus at midgestation (15.8 +/- 6.0 ng/g tissue) was not altered in fetuses whose mothers had been treated with estradiol (17.6 +/- 0.9 ng/g). Hypothalamic CRH concentrations in fetal baboons of late gestation (20.7 ng/g; n = 2) were also similar to mean CRH values measured at midgestation but, owing to the marked increase in weight of the fetal hypothalamus with advancing pregnancy, the content of hypothalamic CRH in late gestation (28.8 ng/structure) exceeded (p < 0.01) that at midgestation. Mean levels of CRH mRNA at midgestation when expressed per cell (17.4 +/- 1.3 grains per cell) or per unit area of PVN (375 +/- 20 grains per area) were similar to respective values in late gestation (18.3 +/- 1.1 grains per cell; 350 +/- 55 grains per area; n = 3 per group). These findings support the suggestion that the increase in fetal pituitary POMC mRNA expression and ACTH peptide previously reported to occur normally between midgestation and term are not associated with a concomitant increase in hypothalamic CRH peptide or CRH mRNA concentrations. Moreover, it would appear that by midgestation, hypothalamic CRH is available in adequate concentrations to "drive" the fetal pituitary and that it is the levels of maternal cortisol arriving within the fetal circulation, as dictated by estrogen-regulated placental 11 beta-HSD-oxidase activity, that establish the extent to which the fetal pituitary responds to CRH.

Activation of the baboon fetal pituitary-adrenocortical axis at midgestation by estrogen: enhancement of fetal pituitary proopiomelanocortin messenger ribonucleic acid expression.

We have proposed that estrogen, via regulation of placental metabolism of maternal cortisol, regulates the baboon fetal hypothalamic-pituitary-adrenal axis and the timing of the onset of de novo cortisol production. In support of this hypothesis, we demonstrated that the ontogenesis of fetal adrenal steroidogenic enzymes near term could be induced at midgestation by maternal estrogen treatment. In the present study, we determined whether maturation of the fetal adrenal near term and at midgestation after estrogen treatment reflects enhanced expression of the messenger RNA (mRNA) for the ACTH precursor molecule POMC. Fetal pituitaries were obtained on day 100 (n = 7) and day 165 (n = 5) of gestation (term = day 184) from untreated baboons and on day 100 after maternal treatment with estradiolbenzoate (sc; days 70-100; n = 6). Sections were fixed in paraformaldehyde and hybridized with saturating concentrations of an antisense (or sense) oligodeoxynucleotide complementary to bases 297-326 of human POMC mRNA that was 3' end-labeled with [35S]dATP. After stringent washes, sections were placed against Kodak X-Omat film (Eastman Kodak, Rochester, NY) and then dipped in Kodak NTB-2, developed, and counterstained. POMC mRNA (antisense minus sense) was quantified by densitometry and image analysis of silver grains. Specificity of labeling was documented by selective distribution of grains over a dispersed population of cells in sections of anterior pituitary hybridized with antisense, the relative absence of grains in sections incubated with sense, and the absence of grains in neurohypophyseal sections incubated with antisense. Moreover, silver grains were not visible when sections were pretreated with excess radioinert probe. The mean (+/- SE) maternal serum estradiol concentration in baboons treated with estradiol benzoate at midgestation (2.9 +/- 0.4 ng/ml) was greater (P < 0.05) than that in untreated baboons on day 100 (1.0 +/- 0.3) but not significantly different from that in late gestation (1.9 +/- 0.3). In umbilical serum, estradiol concentrations were greater (P < 0.05) at term (3.7 +/- 0.9) than at midgestation (0.7 +/- 0.2) but, unlike maternal values, were not significantly increased at midgestation after treatment of the mother with estradiol (1.1 +/- 0.2). Based on densitometric analysis, mean (+/- SE) pituitary POMC mRNA (absorbance units) was greater (P < 0.05) in baboon fetuses at term (0.57 +/- 0.05) than at midgestation (0.28 +/- 0.03) and increased (P < 0.05) on day 100 (0.43 +/- 0.04) in estrogen-treated animals. Similar results were obtained when data were analyzed as the number of silver grains/0.025 mm2.(ABSTRACT TRUNCATED AT 400 WORDS)

Controlled ovarian hyperstimulation: an adjunct to assisted reproductive technology.

Controlled ovarian hyperstimulation was given to a study group consisting of male factor, endometriosis, and unexplained infertility. The aim of the study was to determine whether COH might be of value for such couples in a setting lacking ART facilities. When compared with their own spontaneous cycles or with a control group (untreated but scanned), COH proved significantly better for unexplained infertility. However, COH was not significantly effective for male factor infertility. This study shows that COH should be offered routinely in general hospitals to couples on long-term waiting lists for ART (especially those facing enforced expectant management).

Superovulation and timed intercourse: can it provide a reasonable alternative for those unable to afford assisted conception?

Superovulation was performed prospectively with pure follicle stimulating hormone (FSH) to a group of 224 infertile patients with ovulatory factor (51), male factor (60), mild/moderate endometriosis (24) and unexplained infertility (72). The aim was to produce three or four leading follicles in order to compensate for a 'deficient' factor. Ovulation was induced with human chorionic gonadotrophin (HCG) and monitoring was performed entirely by serial transvaginal ultrasound on alternate cycles up to a maximum of six cycles (1120 treatment cycles) with intervening cycles being used as self-controls (932 rest cycles). A further control group of 56 patients was matched for age, category and duration of infertility and was only scanned serially (336 control cycles). Seventy-four pregnancies were achieved and 54 delivered, giving a cumulative pregnancy rate per couple of 33% and a cumulative take home baby rate of 24% per couple after a maximum of six cycles of treatment. When compared with the rest or control cycles, treatment was significantly effective for ovulatory (P < 0.001), mild/moderate endometriosis (P < 0.01) and unexplained infertility (P < 0.01) but not for male infertility. Furthermore, pregnancy was five times more likely during the first four treatment cycles (P = 0.00006, odds ratio = 5) at the expense of a significant multiple pregnancy rate (18.9%) and mild/moderate ovarian hyperstimulation rate (12%). We conclude that four cycles of superovulation should be routinely offered to couples on waiting lists for assisted conception or to those unable to afford it, in anovulatory, mild/moderate endometriosis and unexplained infertility. These results need confirmation by a prospective multi-centre randomized study.

Developmental regulation of protein kinase-A and -C activities in the baboon fetal adrenal.

We have previously demonstrated that the estrogen-regulated change in transuteroplacental metabolism of cortisol (F) and cortisone (E) from preferential reduction (E to F) at midgestation to oxidation (F to E) near term results in activation of the hypothalamic-pituitary-adrenal axis of the baboon and the ontogenesis of rate-limiting steroidogenic enzymes, culminating in de novo F secretion. It is well established that transcription of messages activated by peptide-mediated binding to membrane receptors can occur via cAMP-dependent protein kinase-A (PKA) and/or phospholipid/calcium-dependent protein kinase-C (PKC). The present study was designed to determine whether basal levels of PKA and PKC in the fetal adrenal are developmentally regulated during baboon gestation and, thus, could provide the mechanism(s) by which activation of the fetal adrenal near term is mediated. Fetal adrenal glands were obtained on day 100 (n = 8) and day 165 (n = 6) of gestation (term = day 184) from untreated baboons and on day 100 after treatment of the mother with estradiol benzoate, injected sc between days 70-100 to increase estrogen production. PKA activity (picomoles of 32P incorporated into kemptide per min/mg protein) was determined by incubation of adrenal cytosol (12,000 x g; 0.3-30 micrograms protein) in reaction mixtures containing 0.25 mM ATP, 1 x 10(6) dpm [lambda-32P]ATP, and 3 micrograms kemptide in the presence or absence of 0.02 mM cAMP. PKC activity (picomoles of 32P incorporated into histone IIIS per min/mg protein) was determined in cytosol (105,000 x g) and detergent-solubilized membrane fractions after incubation with 0.02 mM ATP, 50 micrograms histone IIIS, and 1 x 10(6) dpm [lambda-32P]ATP in the presence or absence of calcium and phospholipids. Mean (+/- SE) maternal serum estradiol concentrations (nanograms per ml) were 3-fold greater (P < 0.05) at term (1.9 +/- 0.3) than at midgestation and increased (P < 0.05) after treatment with estradiol. PKA activity was greater at term (3965 +/- 546) than at midgestation (2130 +/- 240) and increased (P < 0.05) 2-fold after treatment with estrogen (3525 +/- 416) at midgestation. PKC activity was always 3- to 4-fold lower than that of PKA and was similar in the cytosol and membrane fractions of the cell. In contrast to PKA, cytosolic PKC activity was similar at mid (265 +/- 98)-and late (353 +/- 99) gestation and was not altered by treatment with estradiol (223 +/- 27).(ABSTRACT TRUNCATED AT 400 WORDS)

GIFT and IUI in the district general hospital.

Recent reports on gamete intra-Fallopian transfer (GIFT) in hospitals without IVF support have been encouraging. We report on the use of GIFT and intrauterine insemination (IUI) at Northampton General Hospital, where capital costs for setting up the programme have been low. To date seven IUI and three GIFT treatments have been completed, resulting in three IUI positive pregnancy tests. One miscarried after several positive beta-HCGs and one GIFT pregnancy was achieved.

Medical dilatation of the non-pregnant cervix: the effect of ethinyl oestradiol on the visibility of the transformation zone.

The cervical transformation zone (TZ) was not fully visible in 25 women referred to the colposcopy clinic with cytological suspicion of cervical intraepithelial neoplasia (CIN). Each was given a 5-day course of oral ethinyl oestradiol during the follicular phase of the menstrual cycle, and colposcopy was then repeated. The transformation zone was visible at this second examination in 16 of the 25 patients. Of these patients, 11 were treated with a local destructive technique, nine by laser, two by cryocautery; one patient had a cone biopsy, and four patients did not require treatment. Although cone biopsy was necessary in 8 of the remaining 9 patients it is encouraging that the use of a simple medical regimen allowed us to avoid conisation and its morbidity in almost two thirds of these patients.

Kinetics of killing Listeria monocytogenes by macrophages: rapid killing accompanying phagocytosis.

The kinetics of bactericidal activity of activated macrophages can be precisely described by a mathematical model in which phagocytosis, killing, digestion, and release of degraded bacterial material are considered to occur continuously. To gain a better understanding of these events, I have determined the period of time between first contact of bacteria with macrophages and the onset of killing. Activated rat peritoneal macrophages were incubated for various times up to 15 min with Listeria monocytogenes previously labeled with 3H-thymidine and the unassociated bacteria removed by two centrifugations through a density interface. Both cell-associated radioactivity and cell-associated viable bacteria, determined as colony forming units after sonication of the cell pellet, increased with time of incubation. However, the specific viability of these bacteria, expressed as the ratio of number of viable bacteria per unit radioactivity declined with time, as an approximate inverse exponential, after a lag period of 2.9 +/- 0.8 min. Evidence is given that other possible causes for this decline in specific viability, other than death of the bacteria, such as preferential ingestion of dead Listeria, clumping of bacteria, variations in autolytic activity, or release of Listericidins are unlikely. I conclude therefore that activated macrophages kill Listeria approximately 3 min after the cell and the bacterium first make contact.

Kinetics of killing Listeria monocytogenes by macrophages: correlation of 3H-DNA release from labeled bacteria and changes in numbers of viable organisms by mathematical model.

Conventional methods of assessing antibacterial activities of macrophages by viable counting are limited by the precision of the statistics and are difficult to interpret quantitatively because of unrestrained extracellular growth of bacteria. An alternative technique based on the release of radioactive DNA from labeled bacteria has been offered as overcoming these drawbacks. To assess it for use with macrophages I have made a correlation with the conventional viable counting method using a mathematical model. Opsonized Listeria monocytogenes labeled with 3H-thymidine were exposed to rat macrophages for periods up to 4 hr. Numbers of viable bacteria determined after sonication increased exponentially in the absence of live cells and this growth rate was progressively inhibited by increasing numbers of macrophages. After a lag period of 30-60 min soluble 3H appeared in the supernatant, the amount increasing with time and numbers of macrophages. To correlate these data I developed a mathematical model that considered that changes in numbers of viable organisms were due to the difference between rates of 1) growth of extracellular bacteria and 2) killing within the macrophage. On the basis of this model curves of best fit to the viable counts data were used to predict the release of radioactivity, assuming that death of a bacterium led to the total release of its label. These predictions and the experimental data agreed well, the lag period of 30-60 min between death of the bacterium and release of radioactivity being consistent with intracellular digestion. Release of soluble radioactivity appears to be an accurate reflection of the number of bacteria killed within the macrophage.