Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages.

Nitric oxide (NO) is produced by numerous different cell types, and it is an important regulator and mediator of many processes including smooth muscle relaxation, neurotransmission, and murine macrophage-mediated cytotoxicity for microbes and tumor cells. Although murine macrophages produce NO readily after activation, human monocytes and tissue macrophages have been reported to produce only low levels of NO in vitro. The purpose of this study was to determine if stimulated human mononuclear phagocytes produce inducible nitric oxide synthase (iNOS) mRNA, protein, and enzymatic activity. By reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we show that human monocytes can be induced to express iNOS mRNA after treatment with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma). By immunofluorescence and immunoblot analyses, we show monocytes and peritoneal macrophages contain detectable levels of iNOS antigen after stimulations with cytokines in vitro. Control monocytes or those cultured with LPS and/or various cytokines have low levels of NOS functional activity as measured by the ability of cell extracts to convert L-arginine to L-citrulline, and they produce low levels of the NO catabolites nitrite and nitrate. Peritoneal macrophages have significantly enhanced nitrite/nitrate production and NOS activity after treatment with LPS and/or IFN-gamma, whereas monocyte nitrite/nitrate production and NOS activity are not altered by the treatments. Monocytes cultured with various live or heat-killed bacteria, fungi, or human immunodeficiency virus (HIV)-1 do not produce high levels of nitrite/nitrate. Antibodies against transforming growth factor-beta (TGF-beta), a factor known to inhibit iNOS expression and NO production in mouse macrophages, do not enhance NO production in human monocytes or macrophages. Biopterin, an obligate cofactor of iNOS enzymatic activity, is undetectable in freshly isolated or cultured human monocytes and peritoneal macrophages. However, replenishment of intracellular levels of tetrahydrobiopterin by culture with the cell-permeable, nontoxic precursor sepiapterin does not enhance the abilities of the human mononuclear phagocytes to produce NO in vitro. Mixing experiments show no evidence of a functional NOS inhibitor in human mononuclear phagocytes. Thus, we demonstrate that human mononuclear phagocytes can produce iNOS mRNA and protein, and (despite this) their abilities to generate NO are very low.

Differential effects of interleukin-1 alpha, tumor necrosis factor-alpha, indomethacin, hydrocortisone, and macrophage co-culture on the proliferation of human fibroblasts and peritoneal mesothelial cells.

OBJECTIVE: We sought to determine whether human fibroblasts and peritoneal mesothelial cells (PMC) are under comparable proliferative controls. METHODS: Human PMC and human fibroblasts were obtained from primary culture of excised explants from infertile women. The proliferation of PMC, as determined by tritiated thymidine incorporation, was compared with that of fibroblasts in the presence of human peritoneal macrophages, interleukin-1 alpha (IL-1), tumor necrosis factor-alpha (TNF), indomethacin, and hydrocortisone. Data were analyzed by one-way and multifactorial analyses of variance, with Bonferroni adjustments for multiple comparisons. RESULTS: Disparate proliferation was observed between fibroblasts and mesothelial cells with the additives studied. Proliferation of fibroblasts was inhibited (P < .001) when co-cultured with macrophages, IL-1, and TNF. Indomethacin and hydrocortisone overcame the inhibitory effects of macrophage co-culture. By contrast, PMC increased proliferation when cultured with macrophages (P < .001) but were unaffected by IL-1 or TNF and were not altered when indomethacin or hydrocortisone was added to the macrophage co-culture. CONCLUSION: Human PMC and fibroblasts differentially proliferate in response to putative regulatory controls. This suggests that these cells, which play critical roles in peritoneal wound repair, should be considered separately in developing medical strategies to prevent postsurgical adhesions.

Vaginal administration of low-dose conjugated estrogens: systemic absorption and effects on the endometrium.

OBJECTIVE: To test the hypothesis tha a very-low-dose regimen of vaginal estrogen would provide effective relief from atrophic vaginitis without endometrial proliferation. METHODS: Twenty postmenopausal women with symptoms, signs, and cytologic evidence of atrophic vaginitis were enrolled. Each subject was treated with 0.3 mg of conjugated estrogens, administered vaginally 3 nights per week for 6 months. We examined the following outcomes: symptoms, vaginal cellular (cytologic) maturity, endometrial histology, sonographic evaluation of endometrial thickness, Doppler measures of uterine artery blood flow, and serum levels of estrone and estradiol. Pre- and post-treatment data were compared for each subject. RESULTS: Satisfactory relief of symptoms occurred in 19 of 20 cases. Vaginal cellular maturation improved significantly with therapy (P < .01). There were no significant changes in endometrial thickness, uterine artery blood flow, or serum estrogen levels. Endometrial proliferation was observed in one case. CONCLUSIONS: Relief from atrophic vaginitis can be achieved with 0.3 mg of conjugated estrogens administered vaginally three times per week. Endometrial proliferation may occur at this low dose, albeit rarely.

The luteal phase in polycystic ovary syndrome during ovulation induction with human menopausal gonadotropin with and without leuprolide acetate.

Little data exist on the effects of adjunctive therapy with leuprolide acetate (LA) in the luteal phase of women with polycystic ovary syndrome (PCOS) undergoing ovulation induction with human menopausal gonadotropin (hMG). Additionally, it is not known whether gonadal steroid concentrations in the luteal phase of induced cycles in PCOS are predictive of pregnancy. In this prospective, randomized study comparing cycles using hMG alone (n = 26) with cycles using hMG with LA (n = 33), no differences were noted between treatment groups in progesterone (P), estradiol (E2), and P:E2 ratios on luteal days 3, 6, and 9. When all treatment cycles were pooled, there were no differences in P, E2, or P:E2 ratios, comparing conception and nonconception cycles. We conclude that adjunctive therapy with LA in PCOS patients undergoing ovulation induction with hMG does not alter the luteal phase concentrations of P, E2, and P:E2. Furthermore, no correlation was found between the serum concentrations of these luteal phase steroids and cycle fecundity.

Conservative management of cervical pregnancy with subsequent fertility.

The reported incidence of cervical pregnancies with subsequent fertility is extremely low. We report a case managed conservatively that allowed for future fertility, and ultimately the delivery of a viable infant at term. The conservative management and a review of the literature are discussed.