Alterations in sensitivity to nonspecific cell-mediated lysis associated with tumor progression: characterization of activated macrophage- and natural killer cell-resistant tumor variants.

Certain "membrane-mutant," lectin-resistant (Lecr) variants derived from the highly metastatic and poorly immunogenic DBA/2 mouse tumor MDAY-D2 previously were found to differ substantially in their ability to grow and to metastasize. In the present study, the parental MDAY-D2 tumor and several wheat germ agglutinin-resistant (WGAr) variants were examined for alterations in sensitivity to activated macrophage (M phi)- and natural killer cell (NK)-mediated lysis. The results indicated that selection in WGA after mutagenic treatment of a metastatic parental tumor cell line (MDAY-D2), which was M phi-sensitive (M phi S) and NK-resistant (NKR), can result in the isolation of a significantly M phi-resistant (M phi R) and NK-sensitive (NKS) tumor variant, MDW4. The in vivo hybridization of the M phi R, NKS, Lecr MDW4 variant with a normal host-derived cell within a primary subcutaneous tumor, previously demonstrated to result in the progressive and selective outgrowth and metastasis of hybrid products, was found to be associated directly with reversion to the M phi S, NKR phenotype of the metastatic parental MDAY-D2 cell line. DMA/2 mice given iv injections of 10(5) M phi R, NKS cells (MDW4 or MDW4-110c1, a cloned line isolated from a subcutaneous primary tumor of an MDW4-injected animal) survived for a significantly prolonged period as compared to animals given injections of either the parental tumor or M phi S, NKR hybrid products isolated from a MDW4 subcutaneous primary tumor (MDW4-110c2) or visceral metastases (MDW4-24a, MDW4-24b, and MDW4-24c). The results clearly indicate an inverse relationship among the tumor variants in their ability to be lysed by either M phi or NK and suggest a central role for NK rather than M phi surveillance in this tumor system.

The zona reticularis is the site of biosynthesis of dehydroepiandrosterone and dehydroepiandrosterone sulfate in the adult human adrenal cortex resulting from its low expression of 3 beta-hydroxysteroid dehydrogenase.

Based on indirect evidence, it has often been assumed that the zona reticularis of the adult human adrenal cortex is the source of the adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS), but direct tests of this concept have been few. Using the techniques of cell culture, Northern blotting, and RIA, we compared the properties of separated adult zonal cells to those of fetal zone cells, a cell type well known to secrete large amounts of DHEA(S) due to its low expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD). In nine glands from donors of a wide age range, the zona fasciculata and zona reticularis were separated and dissociated, and the cells were placed in culture. After 5 days, serum was removed by a 24-h period in serum-free defined medium followed by a 24-h exposure to cAMP analogs, with the optional addition of insulin, also in serum-free medium. The separated fasciculata and reticularis cells showed large differences in the DHEA(S)/cortisol (F) production ratios from added pregnenolone precursor, consistent with the synthesis of only F and essentially no DHEA(S) by fasciculata cells and with the synthesis of mostly DHEA(S) with little or no F by both reticularis cells and fetal zone cells. The different patterns of steroidogenesis were accompanied by a much lower level of expression of type II 3 beta HSD in reticularis cells, similar to that in fetal zone cells. In contrast, other genes were similarly regulated in the two adult zones and in the fetal zone by both cAMP and insulin. The levels of messenger ribonucleic acids for 17 alpha-hydroxylase, cholesterol side-chain cleavage enzyme, 21-hydroxylase, and 11 beta-hydroxylase responded to cAMP and insulin in both reticularis cells and fetal zone cells in the same pattern as that previously established in fasciculata cells. The central role of the limited expression of 3 beta HSD in the DHEA(S)-synthesizing property of reticularis cells was established by inhibition of 3 beta HSD in fasciculata cells with trilostane, which caused them to increase their DHEA/F production ratio to a level exceeding even that in fetal zone cells. There did not appear to any age-related changes in gene expression that could account for the large age-related decline in DHEA(S) biosynthesis in humans in either reticularis or fasciculata cells. Thus, the most likely cause of the age-related decline in adrenal androgen biosynthesis is an age-related decline in the number of functional reticularis cells, without a major change in the differentiated properties of the zonal cells as a function of age.

Differentially expressed genes in zona reticularis cells of the human adrenal cortex.

The zona reticularis (ZR) cell in the human adrenal cortex is responsible for the secretion of dehydroepiandrosterone, but its biology, origin, and putative decrease in number during aging are poorly understood. In the present experiments, we investigated to what extent ZR and zona fasciculata (ZF) cells differ in patterns of gene expression. Both cell types were purified by microdissection from adult adrenal cortex specimens. After a brief period in culture, RNA was harvested from the cells and used to prepare radioactively labeled probes following amplification by PCR. Probes were used in hybridizations of arrays of cDNAs on nylon membranes (PCR products or plasmids obtained from an adrenal cDNA library). Analysis of hybridization intensities showed that 17 of the 750 genes studied differed in expression by more than 2-fold. Several genes expressed at higher levels in ZR cells encode components of the major histocompatibility complex or enzymes involved in peroxide metabolism. Members of the tubulin gene family were expressed at higher levels in ZF cells. Differential expression of four of the genes was confirmed by Northern blotting. These differences show that although ZR and ZF cells are similar in gene expression, ZR cells have a gene expression pattern related to the unique biology of this cell type.

Mechanisms of insulin inhibition of ACTH-stimulated steroid secretion by cultured bovine adrenocortical cells.

Results of previous studies indicated that insulin at levels comparable to those in humans during hyperinsulinemia decreased ACTH-stimulated cortisol and androstenedione secretion by bovine adrenal fasciculata-reticularis cells in primary culture. In the present studies this inhibitory action was examined further by comparing the effects of insulin on ACTH-stimulated corticosteroid secretion with its effects on 8-(4-chlorophenylthio)-cAMP (cpt-cAMP), forskolin- and [5val]angiotensin II (Ang II)-stimulated corticosteroid secretion. Effects on corticosteroid secretion were correlated with effects on cAMP accumulation and rates of cAMP production. Monolayers were incubated for 24 h in the absence or presence of each agonist alone or in combination with insulin. Insulin (1.7 x 10(-9) or 17.5 x 10(-9) M) caused about a 50% decrease in cortisol and androstenedione secretion in response to ACTH (10(-11) or 10(-8) M). Insulin also decreased ACTH-stimulated aldosterone secretion by cultured glomerulosa cells. Cpt-cAMP (10(-4) or 10(-3) M)-stimulated increases in cortisol and androstenedione secretion were inhibited by insulin, but to a lesser extent than those in response to ACTH. The inhibition of cpt-cAMP-stimulated steroid secretion was not related to increased degradation of the cyclic nucleotide. Increases in cortisol and androstenedione secretion caused by a submaximal concentration (10(-6) M) of forskolin were decreased 50-70% by insulin. In contrast, insulin failed to significantly affect cortisol or androstenedione secretion caused by a maximal concentration (10(-5) M) of forskolin. The secretory responses to Ang II (10(-8) M) were also unaffected by insulin. The effect of insulin to inhibit ACTH-stimulated steroid secretion was accompanied by a reduction in cAMP accumulation as well as an apparent inhibition of adenylate cyclase activation. These data indicate that the effect of insulin to attenuate ACTH-stimulated corticosteroid secretion results from both an inhibition of ACTH-stimulated adenylate cyclase activity and an antagonism of the intracellular actions of cAMP.

Effect of postmenopausal estrogen replacement on circulating androgens.

OBJECTIVE: To determine the effect of estrogen replacement therapy (ERT) on serum androgen levels in postmenopausal women. METHODS: We measured serum dehydroepiandrosterone (DHEA), DHEA-sulfate, testosterone, estradiol (E2), LH, FSH, and sex hormone binding globulin in 8:00 AM fasting serum samples from a previous randomized, blinded, placebo-controlled crossover study in which 28 postmenopausal women (27 naturally menopausal) were given 2 mg/day of oral micronized estradiol. The treatment arms were 12 weeks with a 6-week washout. RESULTS: Estrogen replacement therapy raised mean (+/- standard error of the mean [SEM]) serum E2 from 8.7 +/- 1.0 to 117 +/- 18.7 pg/mL (P < .001 from baseline). Concurrently, mean (+/- SEM) DHEA-sulfate fell from 67.3 +/- 9.6 to 52.1 +/- 6.4 micrograms/dL (P < .001), and mean (+/- SEM) testosterone fell from 16.1 +/- 2.4 to 9.4 +/- 1.4 ng/dL (P = .006). Both FSH and LH declined significantly. Sex hormone binding globulin increased by 160% with ERT (P < .001). CONCLUSION: Menopausal ERT decreases serum androgen levels, decreasing DHEA-sulfate and testosterone by 23% and 42%, respectively. Whereas the decline in testosterone is likely due to decreased LH-driven ovarian stromal steroidogenesis, the declining levels of DHEA-sulfate also may imply a direct adrenal effect of estrogen. Bioavailable testosterone likely is reduced even more profoundly because sex hormone binding globulin is increased 160% by estrogen. Thus, menopausal ERT may induce relative ovarian and adrenal androgen deficiency, creating a rationale for concurrent physiologic androgen replacement.

Ovarian hyperstimulation augments adrenal dehydroepiandrosterone sulfate secretion.

OBJECTIVE: To determine if factor(s) secreted by the ovaries during hyperstimulation potentiate basal and ACTH-stimulated adrenal androgen secretion. DESIGN: Retrospective and prospective and prospective clinical study. SETTING: University tertiary care center infertility clinic. PARTICIPANTS: Two hundred thirteen hyperstimulation cycles in endocrinologically normal women were identified from 92 patients with ovulatory infertility, aged 25 to 45 years. Further, seven endocrinologically normal infertile women, aged 22 to 37 years, who were undergoing empiric ovarian hyperstimulation for infertility were identified and studied. INTERVENTIONS: In the previously performed cycles, basal and peak serum DHEAS and cortisol (F) levels were assayed and compared with each other and to the extant E2 levels. Additionally, at the baseline and the peak of ovarian hyperstimulation cycles, a standard ACTH test was performed and serum was assayed for DHEAS, DHEA, and F. MAIN OUTCOME MEASURE: Basal and ACTH-stimulated serum DHEAS, DHEA (prospective part only), and F concentrations. Where applicable, mean peak values were generated and compared between the baseline and the peak of stimulation with or without a correction for intrapatient variability in F secretion. RESULTS: Basal serum DHEAS levels rose with ovarian hyperstimulation independent of F. Post-ACTH mean peak value concentrations rose with ovarian hyperstimulation for DHEAS but not DHEA or F. CONCLUSIONS: Ovarian hyperstimulation potentiates basal and ACTH perturbed adrenal DHEAS secretion. This implies the existence of a humoral ovarian factor(s) that mediate this ovarian-adrenal cross-talk.

Replacement of dehydroepiandrosterone enhances T-lymphocyte insulin binding in postmenopausal women.

OBJECTIVE: To demonstrate bioavailability of 3 weeks of oral micronized DHEA and to delineate changes induced on insulin sensitivity, morphometric indexes, and lipoprotein profiles. DESIGN: Oral micronized DHEa (50 mg/d) was administered in 3-week treatments to 11 postmenopausal women in a prospective, placebo-controlled, randomized, blinded, crossover trial with an interarm washout. After dose (23 hour) serum DHEA, DHEAS, T, and cortisol levels were measured, as were fasting lipoproteins, oral glucose tolerance tests (OGTT), T-lymphocyte insulin binding and degradation, and urine collagen cross-links. Morphometric changes were determined by hydrostatic weighing. RESULTS: Dehydroepiandrosterone sulfate, DHEA, T, and free T increased up to two times premenopausal levels with treatment. Fasting triglycerides declined; no change in collagen cross-links or morphometric indexes was noted. Oral glucose tolerance test parameters did not change, but both T-lymphocyte insulin binding and degradation increased with DHEA. CONCLUSION: Fifty milligrams per day of oral DHEA gives suprahysiologic androgen levels; 25 mg/d may be more appropriate. Dehydroepiandrosterone enhanced tissue insulin sensitivity and lowered serum triglycerides. Rationale is provided for postmenopausal replacement therapy with this androgen.

Fertilization after standard in vitro fertilization versus intracytoplasmic sperm injection in subfertile males using sibling oocytes.

OBJECTIVE: To compare conventional IVF with ICSI in the subfertile male population using sibling oocytes. Results from males with isolated severe teratozoospermia also are analyzed. DESIGN: Prospective experimental study. SETTING: University based IVF clinic. PATIENT(S): Group A: 18 patients with one or more abnormalities in count, motility, or morphology. Group B: 20 patients with isolated severe teratozoospermia (< or = 4% Kruger Strict Criteria). INTERVENTION(S): Ovulation induction, random allocation of sibling oocytes, and IVF or ICSI. MAIN OUTCOME MEASURE(S): Fertilization rates (fertilization per cycle, fertilization per oocytes, and fertilization per couple) and embryo quality. RESULT(S): In group A, fertilization occurred in 13 of 18 (72%) of IVF cycles and 17 of 18 (94%) of ICSI cycles. Overall, 69 of 120 (58%) oocytes fertilized after IVF, whereas 80 of 131 (61%) fertilized after ICSI. The mean (+/-SEM) percent of oocytes fertilized per couple was 44.6%+/-9.0% with IVF and 62.7%+/-5.6% with ICSI (not statistically significant). In group B, fertilization occurred in 18 of 20 (90%) cycles after IVF and 20 of 20 (100%) cycles with ICSI. Overall, 54 of 113 (48%) of the oocytes fertilized after IVF, whereas 82 of 124 (66%) fertilized with ICSI. The mean (+/-SEM) percent of oocytes fertilized per couple was 50.9%+/-7.1 % with IVF and 66.6%+/-4.7% with ICSI. No statistically significant difference in embryo quality after IVF versus ICSI was demonstrated. CONCLUSION(S): With severe teratozoospermia, ICSI results in higher fertilization rates than conventional IVF, without altering embryo quality. In our subfertile male population, there is a trend toward improved fertilization with ICSI, with less failed fertilization.

Postmenopausal dehydroepiandrosterone administration increases free insulin-like growth factor-I and decreases high-density lipoprotein: a six-month trial.

OBJECTIVE: To determine the effect of administering 6 months of oral postmenopausal DHEA therapy on serum DHEA, DHEAS, and T levels and on physiologic endpoints including lipoproteins and insulin-like growth factor-I (IGF-I). DESIGN: Randomized, double-blind, parallel trial. SETTING: Academic referral practice. PATIENT(S): Thirteen normal-weight or overweight, healthy, nonsmoking, postmenopausal women. INTERVENTION(S): Administration of oral micronized DHEA (25 mg/d). MAIN OUTCOME MEASURE(S): Monthly fasting 23 hours postdose levels of serum DHEA, DHEAS, T, lipoproteins, IGF-I, IGF binding protein-3 (IGFBP-3), and liver function tests. Morphometric indices by dual-energy x-ray absorptiometry scan (percent body fat; lean body mass), immune indices, and insulin sensitivity. RESULT(S): Levels of DHEA, DHEAS, and T all rose into premenopausal ranges, but after 6 months, levels of DHEA and T did not differ from baseline or placebo. At 3 months, the ratio of IGF-I to IGFBP-3 rose by 36.1% +/- 12.7%, but it fell to placebo values by 6 months. High-density lipoprotein and apolipoprotein A1 levels declined. CONCLUSION(S): Patients appeared to tolerate 6 months of DHEA therapy well. Given the small study size, no statistically significant differences in morphometric indices, immune indices, or insulin-sensitizing properties were observed, but significant attenuation of bioavailability occurred. Supplementation with DHEA increased IGF-I/IGFBP-3 levels at 3 months and decreased high-density lipoprotein and apolipoprotein A1 levels at 6 months.

Heterotopic abdominal pregnancy treated at laparoscopy.

OBJECTIVE: To report a case of laparoscopic treatment of a heterotopic primary abdominal pregnancy after IVF with preservation of the concurrent intrauterine pregnancy. DESIGN: Case report. SETTING: University-based IVF program. PATIENT(S): A woman with a heterotopic abdominal pregnancy after IVF-ET. INTERVENTION(S): Pituitary down-regulation with luteal leuprolide acetate, ovulation induction with menotropins, IVF-ET, progesterone in oil for luteal support, laparoscopy, and resection of the abdominal gestation. MAIN OUTCOME MEASURE(S): Human chorionic gonadotropin levels, pelvic ultrasound examinations, and laparoscopic and pathologic findings. RESULT(S): A heterotopic abdominal pregnancy occurred after IVF-ET and was treated successfully with laparoscopy. The concurrent intrauterine pregnancy was delivered at term. CONCLUSION(S): Early diagnosis of an ectopic abdominal pregnancy allowed successful laparoscopic treatment, without sequelae to the intrauterine gestation.

Induction of steroidogenic enzyme genes by insulin and IGF-I in cultured adult human adrenocortical cells.

Insulin and the insulin-like growth factors (IGFs) have multiple role in gene expression in steroidogenic cells. We investigated the regulation of steroidogenic enzyme gene expression by insulin and IGF-I in primary cultures of human adrenocortical cells from donors of ages 19-77 years. The effects of insulin and IGF-I observed here were independent of age and sex of the donor. After 5 days in serum-containing medium, cultures were exposed to insulin or IGF-I together with cyclic AMP analogs or ACTH in serum-free defined medium. Insulin and IGF-I at physiological concentrations increased mRNA levels for 17 alpha-hydroxylase and type II 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in the absence of cyclic AMP or ACTH. They had lesser effects on 21-hydroxylase and cholesterol side-chain cleavage enzyme mRNA levels and were3 without effect on 11 beta-hydroxylase mRNA. All steroidogenic enzyme mRNAs were strongly increased by cyclic AMP or ACTH, and this increase was potentiated by insulin or IGF-I. These effects of insulin and IGF-I were accompanied by decreases in the ratio of dehydroepiandrosterone/cortisol synthesized from pregnenolone by the cultures. Induction of steroidogenic enzyme genes in adult human adrenocortical cells by insulin and IGF-I is unlikely to occur by means of a cyclic AMP-dependent mechanism. These data increase the evidence for an important regulation of steroidogenesis by these hormones.

Frey's syndrome: a preventable phenomenon.

Gustatory sweating, or Frey's syndrome, is a fairly common sequela of partial or radical parotidectomy, submaxillary gland surgery, or radical neck dissection. It is caused by an anastomotic communication with facial sweat glands by parasympathetic secretomotor nerve fibers intended for the excised parotid gland; treatments, whether surgical or topical, generally have been less than satisfactory. We present the first documented prophylactic approach to Frey's syndrome that is performed during and as part of parotidectomy. The surgery involves use of the superficial aponeurotic system (SMAS) as an interposing flap to interrupt the anastomotic nerve communication with the sweat glands. The SMAS is derived from the fascia in the periauricular cheek and neck area that is continuous with the platysma muscle. In a prospective study in 55 patients undergoing elective parotidectomy, the SMAS flap was elevated during the beginning of the operative procedure once it had been determined that fashioning of the flap would in no way compromise tumor excision. In all cases, at follow-up, there has been no clinical evidence of development of Frey's syndrome. We have shown that the development of the SMAS flap in parotid gland resections is an effective new approach both as a preventative measure against Frey's syndrome and as an aesthetic improvement over the usual defect typical of parotidectomies.

Androgen replacement therapy in women: myths and realities.

In recent years, much attention has been directed at the potential of androgen replacement in the menopausal woman. Testosterone (T) replacement, in various forms, is widely used. However, evidence is lacking for a profound T deficiency state with natural menopause. Data confirming efficacy are also scant, and side effects have been demonstrated with prolonged therapy. The adrenal androgens, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S), also in contradistinction to T, decline substantially with age. Preliminary studies involving replacement of physiologic levels of DHEA have demonstrated some potential benefits: enhancement of the immune system and enhancement of the growth hormone axis. However, long-term trials have not been performed to date, so this modality of androgen replacement remains in the realm of clinical investigation. Ovarian and adrenal androgen replacement in menopausal women, while theoretically appealing, remains imperfect to date and should be used judiciously, if at all.

Reconstruction of defects following Mohs' surgery.

Repairs of surgically produced wounds require fore-thoughts and planning individualized for particular patients and particular original conditions. Nowhere is this more important than in patients treated by Mohs' methods for cutaneous cancers. Indications for early and late repair and methodology by a variety of grafts and flaps are explored and illustrated.

Testosterone delivery systems for women: present status and future promise.

In women, testosterone (T) is increasingly recognized as a steroid with multiple non-reproductive effects. Testosterone deficiency in menopausal women is more common than appreciated, particularly in patients on hormone replacement or with surgical menopause. Replacement of T is an established therapy for male hypogonadism, and as a result innovative new delivery systems have evolved to optimize physiologic delivery. However, in women, modalities of T replacement remain underdeveloped and at present provide artificial and/or supraphysiologic androgen levels. This review discusses the androgen replacement modalities presently available for women, and those being developed for future use.

Changes in adrenocortical function with aging and therapeutic implications.

Throughout life, the adrenal cortex exhibits dramatic morphogenic and steroidogenic changes. While there is subtle senescent decline in aldosterone, and a similarly subtle increase in cortisol, the adrenal androgens dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) decline with age in a situation similar to menopause, and this decline is considered by some to aggravate some age-related diseases. This decline is associated with an almost complete loss of the inner zone of the adrenal cortex, known as the zona reticularis. This review addresses these adrenal cortical changes, and explores their clinical significance. In particular, the clinical data on DHEA replacement in aging is addressed.