Medical records documentation of constipation preceding Parkinson disease: A case-control study.

OBJECTIVE: Parkinson disease (PD) may affect the autonomic nervous system and may cause constipation; however, few studies have explored constipation preceding the motor onset of PD. We investigated constipation preceding PD using a case-control study design in a population-based sample. METHODS: Using the medical records-linkage system of the Rochester Epidemiology Project, we identified 196 subjects who developed PD in Olmsted County, MN, from 1976 through 1995. Each incident case was matched by age (+/-1 year) and sex to a general population control. We reviewed the complete medical records of cases and controls in the medical records-linkage system to ascertain the occurrence of constipation preceding the onset of PD (or index year). RESULTS: Constipation preceding PD or the index year was more common in cases than in controls (odds ratio [OR] 2.48; 95% confidence interval [CI] 1.49 to 4.11; p = 0.0005). This association remained significant after adjusting for smoking and coffee consumption (ever vs never), and after excluding constipation possibly induced by drugs. In addition, the association remained significant in analyses restricted to constipation documented 20 or more years before the onset of motor symptoms of PD. Although the association was stronger in women than in men and in patients with PD with rest tremor compared with patients with PD without rest tremor, these differences were not significant. CONCLUSIONS: Our findings suggest that constipation occurring as early as 20 or more years before the onset of motor symptoms is associated with an increased risk of Parkinson disease.

Anemia or low hemoglobin levels preceding Parkinson disease: a case-control study.

OBJECTIVE: It has been suggested that anemia may be a risk factor for dementia, for restless legs syndrome, and for Parkinson disease (PD). Thus, we investigated the association of anemia with the subsequent risk of PD using a case-control study design. METHODS: We used the medical records-linkage system of the Rochester Epidemiology Project to identify 196 subjects who developed PD in Olmsted County, Minnesota, from 1976 through 1995. Each incident case was matched by age (+/-1 year) and sex to a general population control. We reviewed the complete medical records of cases and controls in the system to detect anemia defined using the World Health Organization criteria. RESULTS: Anemia was more common in the history of cases than of controls (odds ratio 2.00, 95% confidence interval 1.31-3.06, p = 0.001). The association remained significant after adjustment for cigarette smoking, exposure to pesticides, or hysterectomy (in women). The association was not significantly different between men and women, or between PD patients with or without rest tremor. Analyses stratified by time of onset of anemia showed a greater association for anemia that started 20 to 29 years before the onset of PD. Hemoglobin levels were slightly but consistently lower in cases than in controls across all ages. CONCLUSIONS: Our results support an association between anemia experienced early in life and the later development of Parkinson disease. The interpretation of this association remains uncertain.

Transcriptional activation of indoleamine dioxygenase by interleukin 1 and tumor necrosis factor alpha in interferon-treated epithelial cells.

Interferon (IFN)-gamma-induced indoleamine 2,3-dioxygenase (IDO) activity is enhanced synergistically by interleukin (IL-)1, tumor necrosis factor-alpha (TNF-alpha) and LPS in IFN-treated macrophages by increasing IDO mRNA concentration. These studies demonstrate that IFN-treated HeLa cells also exhibit dose-dependent enhancement of IDO induction by TNF-alpha and IL-1, with maximal effects at concentrations of 5 ng/ml and 3 ng/ml, respectively. Furthermore, with sub-optimal IFN concentrations, cells treated with maximally effective concentrations of TNF-alpha or IL-1alpha required 3-5 times less IFN to induce the same level of IDO activity as that observed with IFN alone. To detect changes in transcriptional activation of the IDO gene, HeLa cells were transfected with a plasmid containing the IDO 5' regulatory region upstream of a green fluorescent protein (GFP) reporter gene. In transfected cells, IFN induced both IDO activity and GFP that was detected by flow cytometry. When cell-sorted, transfected cells were stimulated with IFN in combination with TNF-alpha or IL-1 but not LPS, increased GFP was detected in comparison to transfected cells treated with IFN alone. Furthermore, increases in GFP expression correlated with IDO enzymatic activity, indicating that combinations of IFN with IL-1 or TNF-alpha increase the transcriptional activity of the IDO promoter region.

Tumor necrosis factor-alpha and lipopolysaccharide enhance interferon-induced antichlamydial indoleamine dioxygenase activity independently.

In macrophages, interleukin-1 (IL-1) and lipopolysaccharide (LPS) enhance the antichlamydial effect of interferon-gamma (IFN-gamma) by increasing indoleamine 2,3-dioxygenase (IDO) activity in a dose-dependent manner. Our objectives were to characterize the antichlamydial effect of tumor necrosis factor-alpha (TNF-alpha) on IFN-induced IDO activity and to establish the relationship between LPS and TNF-alpha in IDO potentiation. TNF-alpha inhibited chlamydial growth in a dose-dependent manner only in IFN-treated macrophages. Furthermore, excess tryptophan reversed the effect of combined cytokine treatment, indicating that IDO alone was responsible for chlamydial inhibition. The promonocyte THP-1 cell line, previously used to model the effect of IL-1 on IDO mRNA expression, was treated with IFN-gamma and increasing concentrations of LPS or TNF-alpha. IDO mRNA was quantified by RT-PCR, and IDO activity was measured by HPLC at 24 and 48 h after treatment, respectively. Both LPS and TNF-alpha enhanced IDO activity and IDO mRNA expression, with maximal IDO induction at 100 ng/ml LPS or 5 ng/ml TNF-alpha. Anti-TNF-alpha failed to neutralize the effects of LPS treatment, and insufficient TNF-alpha or IL-1 was produced by LPS-treated THP-1 cells to account for the enhancing effect of LPS, indicating that the effect of LPS on IDO was independent of TNF-alpha and IL-1.

Potentiation of interferon-induced indoleamine 2,3-dioxygenase mRNA in human mononuclear phagocytes by lipopolysaccharide and interleukin-1.

Previous studies have shown that interleukin-1 (IL-1) enhances interferon (IFN)-gamma-induced indoleamine 2,3-dioxygenase (IDO) enzymatic activity in human monocyte-derived macrophages by increasing expression of IDO mRNA. The objectives of this study were to see if IL-1 also enhances IFN-beta-induced IDO activity by increasing specific mRNA expression and to determine if lipopolysaccharide (LPS) enhances IFN-induced IDO activity in a similar manner. Macrophages were treated with combinations of IFN-beta or IFN-gamma as inducer and LPS or IL-1 as potentiator. After 48 h, IDO mRNA expression was assessed by RT-PCR, and IDO activity was determined by HPLC. LPS alone induced IDO mRNA expression and also increased IDO mRNA expression induced by either type of IFN. Furthermore, IL-1 enhanced IFN-beta-induced IDO mRNA expression. When IDO mRNA was assessed 6 h after treatment, mRNA was detected at concentrations of IFNs or potentiator or both in which enzymatic activity at 48 h was undetectable. Thus, although the mechanism of potentiation of IFN-induced IDO by LPS and by IL-1 involves increased expression of IDO mRNA, it appears that temporal differences in IDO mRNA expression are also important.

Interleukin-1 enhances indoleamine 2,3-dioxygenase activity by increasing specific mRNA expression in human mononuclear phagocytes.

The objective of this study was to determine the utility of the THP-1 monocytic leukemia cell line as a model for analyzing molecular mechanisms involved in enhancement of interferon (IFN)-gamma-induced indoleamine dioxygenase (IDO) activity by interleukin-1 (IL-1). Following treatment of THP-1 cells with combinations of IFN-gamma and IL-1, IDO activity and IDO mRNA were quantified by HPLC and radioanalytic imaging of RT-PCR products, respectively. IL-1 increased the amount of IDO activity and the expression of IDO mRNA in IFN-treated cells; IL-1 alone had no effect on untreated THP-1 cells. Because IDO gene regulation might differ between immature THP-1 cells and mature macrophages, experiments were repeated using primary macrophage cultures. IFN-gamma induced IDO activity, and IDO mRNA was expressed in a dose-dependent manner. In the presence of IL-1, 10 times less IFN was required to obtain the same amount of IDO mRNA and IDO activity. Furthermore, IL-1 alone increased IDO mRNA expression. It appears that unlike what was observed in THP-1 cells, IL-1 transcriptionally activates the IDO gene in primary macrophages. However, increases in IDO activity were not observed following treatment with IL-1 alone. Although the THP-1 cell may be used to model cytokine potentiation of IFN-induced IDO activity, some differences in regulation between THP-1 cells and primary macrophage cultures may exist.

Potentiation of interferon-mediated inhibition of Chlamydia infection by interleukin-1 in human macrophage cultures.

One mechanism by which interferons (IFNs) can inhibit chlamydial infection is by the induction of the enzyme indoleamine 2,3-dioxygenase (IDO), which restricts the availability of tryptophan, which is required for chlamydial growth. Other immunomodulating agents, including interleukin-1 (IL-1), can interact synergistically with IFNs, resulting in increased IDO activity in macrophages. The objectives of this study were to establish that IL-1 can enhance IFN-mediated inhibition of chlamydial growth by increasing the amount of IDO activity induced by IFNs and to identify immunomodulatory agents in culture supernatants from chlamydia-infected macrophages that interact synergistically with IFNs in restricting chlamydial growth. Monocyte-derived macrophages were treated with IL-1 combined with gamma IFN (IFN-gamma) or IFN-beta. The ability of treated cells to support the growth of Chlamydia psittaci was directly related to the amount of IDO activity induced; as IDO activity increased, so did inhibition of chlamydial growth. Furthermore, concentrations of IFNs were identified at which little IDO activity was induced and chlamydial growth was permitted yet which in the presence of IL-1 resulted in increased IDO activity and restriction of chlamydial growth. The addition of exogenous tryptophan reversed the effect of combined IFN and IL-1 treatment, indicating that IDO activity induced by combined cytokine treatment was responsible for chlamydial inhibition. Supernatants from chlamydia-infected macrophages were capable of potentiating IDO induction by IFN-gamma and of restricting the growth of C. psittaci. Antibody to IL-1 beta neutralized the potentiating effects of supernatants from chlamydia-infected cells on both IDO induction and chlamydial inhibition. Thus, IL-1 produced in response to chlamydial infection may contribute to the elimination of the infection.

Upregulation of interferon-induced indoleamine 2,3-dioxygenase in human macrophage cultures by lipopolysaccharide, muramyl tripeptide, and interleukin-1.

The tryptophan decyclizing enzyme indoleamine 2,3-dioxygenase (IDO) was induced in human monocyte-derived macrophages (MDM) treated with human recombinant interferon-beta (IFN-beta) or interferon-gamma (IFN-gamma). Treated cells exhibited dose-dependent increases in IDO when assayed 48 hr after treatment. Cells exposed to IFN-gamma were observed to exhibit consistently higher peak levels of IDO when compared with cells incubated in the presence of IFN-beta. When IFN-beta-treated cells were incubated in the presence of specified amounts of bacterial lipopolysaccharide (LPS) or liposome-encapsulated muramyl tripeptide (MTP), peak IDO activity increased such that enzyme activity was comparable to maximal activity observed with IFN-gamma-treated cells. LPS and MTP also upregulated IFN-gamma-mediated IDO activity when suboptimal amounts of IFN-gamma were used. When macrophages were costimulated with various concentrations of human recombinant interleukin 1 alpha (IL-1 alpha), along with either maximum-stimulating amounts of IFN-beta or suboptimal amounts of IFN-gamma, IDO activity was upregulated in a manner similar to results obtained using the microbial products as stimuli. While neither IL-1 alpha or IL-1 beta was detected in culture supernatants from macrophages treated with either LPS or MTP (alone or in combination with IFN), IL-1 alpha was detected in cell lysates of macrophages treated with these upregulators. Although neutralizing antibody to IL-1 alpha abolished the upregulatory effect of exogenous IL-1 alpha, it had no effect on upregulation by LPS or MTP. This suggests that although LPS and MTP may induce production of cell-associated IL-1 alpha, upregulation of IDO activity by these agents is independent of IL-1 alpha production and may be mediated through distinct pathways.

Antiparasitic and antiproliferative effects of indoleamine 2,3-dioxygenase enzyme expression in human fibroblasts.

Studies were carried out to evaluate the proposed role of indoleamine 2,3-dioxygenase (INDO) induction in the antimicrobial and antiproliferative effects of gamma interferon (IFN-gamma) in human fibroblasts. The INDO cDNA coding region was cloned in the pMEP4 expression vector, containing the metallothionein (MTII) promoter in the sense (+ve) or the antisense (-ve) orientation. Human fibroblasts (GM637) stably transfected with the sense construct expressed INDO activity after treatment with CdCl2 or ZnSO4, but cells transfected with the antisense construct did not. The growth of Chlamydia psittaci was strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+ or Zn2+. The inhibition correlated with the level of INDO activity induced and could be reversed by the addition of excess tryptophan to the medium. The growth of Toxoplasma gondii was also strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+. Expression of Cd(2+)-induced INDO activity also inhibited thymidine incorporation and led to cytotoxicity in INDO +ve cells but not in INDO -ve cells. Thus, the induction of INDO activity by IFN-gamma may be an important factor in the antimicrobial and antiproliferative effects of IFN-gamma in human fibroblasts.

Cytokine-mediated indoleamine 2,3-dioxygenase induction in response to Chlamydia infection in human macrophage cultures.

The purpose of this study was to characterize further the events leading to the metabolic degradation of tryptophan in Chlamydia-infected cultures in the absence of added interferon (IFN). Macrophages on coverslips were infected with Chlamydia psittaci, and tryptophan decyclization was determined 24 h later by reverse-phase high-performance liquid chromatography. Tryptophan metabolites cochromatographed with kynurenine and N-formylkynurenine, the end products of tryptophan decyclization by the IFN-inducible enzyme indoleamine 2,3-dioxygenase (IDO). Although chloramphenicol pretreatment completely inhibited chlamydial replication, IDO was stimulated to an extent similar to that in untreated, infected cells. No IDO induction was observed in cells pretreated with cycloheximide even though chlamydial growth was slightly greater than in untreated cells. These results indicate that enhanced tryptophan decyclization was due to induction of IDO. IDO induction was dependent on the size of the chlamydial inoculum. Heat- or UV-inactivated chlamydiae induced significantly less IDO activity than viable chlamydiae. Culture supernatants from Chlamydia-infected macrophages induced IDO activity in a dose-dependent manner, suggesting that a secreted product of infected cells was responsible for IDO induction. A combination of neutralizing antibodies to IFN-alpha and IFN-beta inhibited induction of IDO activity by infected cell culture supernatants. Furthermore, IL-1 enzyme-linked immunosorbent assay results indicated the accumulation of IL-1 beta in the culture medium. Thus, induction of IDO in Chlamydia-infected macrophages reflects the production of cytokines in response to infection and may represent a normal host cell response to control intracellular infection.

IFN-gamma-mediated antimicrobial response. Indoleamine 2,3-dioxygenase-deficient mutant host cells no longer inhibit intracellular Chlamydia spp. or Toxoplasma growth.

The role of indoleamine 2,3-dioxygenase (IDO) in IFN-gamma-mediated inhibition of intracellular parasite growth has been examined previously, although earlier work has been largely correlative. In this study, we defined more completely the role of IDO in the IFN-antimicrobial response. Two mutant cell lines, derived from ME180 cells and exhibiting reduced IDO activity (IR3B6A, IR3B6B) were characterized to determine if they retained the capacity to inhibit intracellular Chlamydia and Toxoplasma growth. Mutant cells treated with IFN-gamma exhibited reduced capacity to suppress pathogen growth. The expression of several IFN-regulated genes also was measured to confirm that the inability to inhibit pathogen growth was because of the lack of IDO. The expression of class II MHC, intracellular adhesion molecule-1, MxA, and P68 kinase genes was induced in the IFN-gamma-treated wild type ME180 cells, but was variable in the mutant cell lines, supporting the hypothesis that IFN-gamma-induced production of IDO is a key IFN-gamma-mediated antimicrobial mechanism.

Effects of interferons beta or gamma on neopterin biosynthesis and tryptophan degradation by human alveolar macrophages in vitro: synergy with lipopolysaccharide.

Interferons can induce neopterin biosynthesis and tryptophan degradation in monocytic cells. Indoleamine 2,3-dioxygenase (IDO), an inducible cellular enzyme, metabolizes tryptophan to N-formyl-L-kynurenine. Tryptophan degradation has been linked to interferon-mediated inhibition of replication by intracellular pathogens and inhibition of cancer cell proliferation. We evaluated the ability of the recombinant human interferons beta ser and gamma to stimulate neopterin production and tryptophan degradation in vitro by alveolar macrophages (AM) obtained from normal volunteers by bronchoalveolar lavage. Additionally, because other biologic response modifiers such as lipopolysaccharide (LPS) can also stimulate monocytic cells to produce increased amounts of neopterin and degrade tryptophan, we evaluated the effects of LPS on interferon-induced neopterin production and tryptophan degradation by AM. Both interferon-gamma (IFN-gamma) and interferon-beta (IFN-beta) induced neopterin production and tryptophan degradation by AM with corresponding inhibition of intracellular replication by Chlamydia psittaci in AM, but IFN-gamma was a more potent inducer of these responses than IFN-beta. LPS enhanced neopterin production and tryptophan degradation by interferon-exposed cells. This effect was particularly evident at lower concentrations of interferon, and LPS synergy was more pronounced with IFN-beta than IFN-gamma. Concentrations of LPS that alone had no stimulatory effect on tryptophan degradation synergistically enhanced the induction of IDO activity by lower concentrations of interferon. These studies suggest that IFN-gamma stimulates human AM to produce neopterin and degrade tryptophan more potently than IFN-beta, and that low concentrations of LPS can synergistically enhance such effects of interferons on tissue macrophage metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Aphid derived sugars in the neotropical sandfly--Lutzomyia peruensis.

It is known that a sugar meal is a prerequisite for the transmission of Leishmania by sandflies in the laboratory. Lutzomyia peruensis the proven vector of Leishmania peruviana, was caught by aspiration from crevices in rocks near Chaute in the Rimac Valley, Peru, cryopreserved and analysed for sugars using HPLC. The major sugars present are glucose and fructose as well as smaller amounts of sucrose, maltose, melibiose, turanose and a trisaccharide, probably raffinose. The results indicate that the major carbohydrate sugar meal of Lutzomyia peruensis is aphid honeydew. This is the first report of such behaviour in Neotropical sandflies.

Effects of combinations of interferon-beta ser and interferon-gamma on interferon-inducible proteins and on the cell cycle.

SKCO 1 human colon carcinoma cells have been shown to be synergistically inhibited in their growth by the combinations of alpha-interferon (IFN-alpha) or beta-interferon (IFN-beta ser) and gamma-interferon (IFN-gamma). To determine if a correlation could be established between this synergistic antiproliferative effect and a synergistic induction in IFN-inducible proteins, or a unique perturbation in the cell cycle, we studied the effects of IFN-beta ser and IFN-gamma, alone and in combination, on 2',5'-oligoadenylate (2-5A) synthetase, indoleamine-2,3-dioxygenase (IDO), a human analogue of the murine Mx protein (p78), and the phases of cell cycle. 2-5A synthetase was maximally induced after a 24-h exposure to both IFN-beta and IFN-gamma. A synergistic enhancement of 2-5A synthetase activity was observed only with low concentrations of each IFN (0.05 ng/ml). IDO activity was induced by IFN-gamma and the combination of IFN-beta ser and IFN-gamma, but not IFN-beta ser alone. The differences in IDO activity between IFN-gamma and the combination, however, were not statistically significant. The p78 protein was induced in a dose-dependent manner by IFN-alpha and IFN-beta ser. IFN-gamma enhanced the expression of p78 induction by IFN-alpha or IFN-beta ser, even at concentrations of IFN-gamma that did not induce the protein when administered as a single agent. The combination of IFN-alpha and IFN-beta ser, which results in an antagonistic antiproliferative effect, also resulted in an antagonistic induction of p78. No changes in the cell cycle were observed following exposure to IFN-beta ser, IFN-gamma, or the combination, and treatment with IFN-gamma did not inhibit the accumulation of cells in G2M caused by colchicine. Thus, the synergistic antiproliferative effect produced by IFN-beta ser and IFN-gamma in SKCO 1 cells could not be correlated with a synergistic enhancement in 2-5A synthetase or IDO activity, or with a perturbation in the cell cycle. In contrast, the combination of IFN-gamma and IFN-alpha or IFN-beta ser synergistically enhanced the expression of p78 protein in these cells.

Beta interferon inhibits Toxoplasma gondii growth in human monocyte-derived macrophages.

Gamma interferon (IFN-gamma) previously has been shown to block the replication of Toxoplasma gondii in fibroblasts by the induction of indoleamine 2,3-dioxygenase (IDO) activity. IFN-beta also is known to induce IDO activity in monocyte-derived macrophages, but its ability to block the growth of T. gondii has not been demonstrated. We found not only that the combination of IFN-beta and lipopolysaccharide induced greater IDO activity in monocyte-derived macrophages than did IFN-beta alone but that this combination also was effective in inhibiting the growth of T. gondii. In addition, the inhibition was reversed by the addition of exogenous tryptophan, thus demonstrating that a mechanism by which IFN-beta inhibited T. gondii replication was by the induction of IDO.

Interferon-induced indoleamine 2,3-dioxygenase activity inhibits Chlamydia psittaci replication in human macrophages.

Interferon-gamma (IFN-gamma) previously has been shown to inhibit the replication of Chlamydia psittaci in epithelial cells by inducing indoleamine 2,3-dioxygenase, the enzyme that decyclizes tryptophan to N-formylkynurenine. The role of indoleamine 2,3-dioxygenase in IFN-mediated inhibition of C. psittaci in human macrophages has now been examined. Peripheral blood monocytes from normal donors were isolated and cultivated 10-14 days to allow differentiation to macrophages. Cells were then treated with either IFN-gamma or IFN-beta for 48 h before infection with sufficient C. psittaci to infect approximately 30% of the cells. Infected cells were incubated 24 h, at which time coverslips were fixed, stained with Giemsa, and examined for development of C. psittaci inclusions by light microscopy. Complete inhibition of inclusion development was observed with IFN-gamma. In the absence of lipopolysaccharide, inhibition of C. psittaci by IFN-beta was variable; however, in the presence of lipopolysaccharide, IFN-beta also completely inhibited C. psittaci replication. The addition of excess tryptophan to the culture medium at the time of infection partially reversed the effect of IFN on the inhibition of C. psittaci growth in a concentration-dependent manner. Indoleamine 2,3-dioxygenase activity was determined by measurement of the concentrations of tryptophan and its metabolites in the culture medium after reversed-phase high-performance liquid chromatography. Significant indoleamine 2,3-dioxygenase activity was observed only in macrophages treated with IFN-gamma or combined IFN-beta plus lipopolysaccharide, and resulted in greater than 50% of available tryptophan being catabolized in a 4-h period.(ABSTRACT TRUNCATED AT 250 WORDS)

Interferons and indoleamine 2,3-dioxygenase: role in antimicrobial and antitumor effects.

Indoleamine 2,3-dioxygenase (IDO) is an interferon (IFN)-induced protein that initiates the metabolism of tryptophan along the kynurenine pathway. Although IDO can be induced by IFN-gamma in many cell types, only mononuclear phagocytes have been shown to be induced to decyclize tryptophan by all three IFN classes. Since tryptophan is an essential amino acid necessary for a variety of metabolic processes, depletion of available tryptophan may be an important mechanism for control of rapidly-dividing microbial pathogens and tumors. The purpose of this review is to present evidence that documents the effects of IFN-induced IDO on prokaryotic and eukaryotic pathogens, as well as on a variety of tumor cell lines.

Long-term effects of gamma interferon on chlamydia-infected host cells: microbicidal activity follows microbistasis.

When human monocyte-derived macrophages or a human uroepithelial cell line (T24 cells) was incubated in the presence of gamma interferon (IFN-gamma) for 24 to 48 h and then infected with Chlamydia psittaci, host cells became activated to restrict intracellular C. psittaci growth. A reversal of this inhibition was observed when infected cells were supplemented with excess exogenous tryptophan at the time of infection or at 1 to 3 days after infection. When IFN-gamma-treated, infected cells were incubated for more extended periods of time before the addition of exogenous tryptophan, no recovery of viable chlamydiae was observed. Neither replacement of the IFN-gamma-containing medium with complete medium supplemented with excess tryptophan nor the addition of excess concentrations of all 20 amino acids with and without essential vitamins contributed to the reversal of IFN-gamma-mediated inhibition of chlamydial growth beyond the time when reversal occurred after the addition of exogenous tryptophan alone. These data provide evidence which indicates that although IFN-gamma treatment of host cells initially results in a microbistatic inhibition of intracellular chlamydial development, longer incubations result in microbicidal activity that is irreversible by modulation of essential nutrient levels.