Absence of keratin 1 restricts the course of infection with lymphocytic choriomeningitis virus strain MX.

A rodent-transmitted enveloped lymphocytic choriomeningitis virus (LCMV) is an RNA virus causing persistent infection. During persistent infection, a unique strain MX of LCMV does not yield infectious virions, therefore it is not able to use a receptor for its dissemination, and spreads by cell-to-cell contacts. Virus can be transported to the neighboring cell by different cellular structures such as tunneling nanotubes or cytonemes. Using q-PCR, immunofluorescence, siRNA and western blot, we show that keratin 1 (K1) is essential for the persistent infection caused by LCMV strain MX, and its absence very effectively slows down the course of infection. In contrast, other LCMV strains, namely Clone 13 and Armstrong, which produce expression of K1, desmosomes in cells expressing K1 (42-MG-BA) but not in cells without K1 expression (NIH/3T3). We conclude that the presence of the virus enhances the K1 expression, while the presence of K1 protein potentiates the viral spread in persistently infected cells. Keywords: lymphocytic choriomeningitis virus; keratin 1; persistent infection; desmosomes; virus transport.

Cell-to-cell transmission of lymphocytic choriomeningitis virus MX strain during persistent infection and its influence on cell migration.

Lymphocytic choriomeningitis virus (LCMV) can establish in its host a persistent infection, without any prominent symptoms. Even during this infection, when the infectious virions are not released, the virus still disseminates effectively. A very effective and fast way of infection of neighboring cells utilized by many viruses is cell-to-cell transmission. Viruses use different ways of cell-to-cell spread through the extracellular space or by intracellular means through different protrusions. We have found that LCMV strain MX may use three different types of cell-to-cell transport. Firstly, similar to vaccinia virus, it can use actin to propel the virus towards the neighboring cell. Secondly, virus can travel through the intracellular space inside the tunneling nanotubes, that connect the cells even at longer distances and thirdly, the virus may travel on the surface of the membrane of different protrusions connecting two cells. We have also proved that the cells infected by MX strain of LCMV migrate faster than the uninfected cells or cells infected with a different LCMV strain. In accordance with faster migration, the infected cells form more lamellipodia with high expression of keratin 1. In this work, we have introduced three types of cell-to-cell transmission utilized by strain MX of LCMV and showed that even if the cells are not in tight connection, the virus forces them to migrate faster to join the nearest cell. As we show in this work, the virus may use more than one strategy to move to another cell, while each strategy can substitute another. These ways of transmission are very fast and effective and may have a serious impact on the host. Moreover, targeting the cell-to-cell spread, by inhibiting for instance GTPase dynamin, could be an effective way of virus elimination. Keywords: lymphocytic choriomeningitis virus; transmission; migration; keratin 1; nucleoprotein.

Lymphocytic choriomeningitis virus: ways to establish and maintain non-cytolytic persistent infection.

Lymphocytic choriomeningitis virus (LCMV) is a prototype virus of the Arenaviridae family that is attracting considerable attention both as an important experimental model system to study acute and persistent viral infections, and as a neglected human pathogen of clinical significance. Notably, LCMV is capable of persisting in an infected host, and escaping the immune system. Here we describe the strategies used by the virus to establish and maintain long-term infection in vitro and/or persistent infection in vivo. We discuss how the viral components (RNA, nucleoprotein, glycoprotein, Z protein) manipulate the host cell machinery to facilitate survival and spread of the virus without disturbing the basal cellular processes. Deep understanding of these strategies is inevitable for the development of approaches towards restricting the virus spread and/or preventing its harmful reactivation. This review summarizes the current status in this area and presents ideas emerging from existing data.

Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2.

Endosialin is a transmembrane glycoprotein selectively expressed in blood vessels and stromal fibroblasts of various human tumours. It has been functionally implicated in angiogenesis, but the factors that control its expression have remained unclear. As insufficient delivery of oxygen is a driving force of angiogenesis in growing tumours, we investigated whether hypoxia regulates endosialin expression. Here, we demonstrate that endosialin gene transcription is induced by hypoxia predominantly through a mechanism involving hypoxia-inducible factor-2 (HIF-2) cooperating with the Ets-1 transcription factor. We show that HIF-2 activates the endosialin promoter both directly, through binding to a hypoxia-response element adjacent to an Ets-binding site in the distal part of the upstream regulatory region, and indirectly, through Ets-1 and its two cognate elements in the proximal promoter. Our data also suggest that the SP1 transcription factor mediates responsiveness of the endosialin promoter to high cell density. These findings elucidate important aspects of endosialin gene regulation and provide a rational frame for future investigations towards better understanding of its biological significance.

Lymphocytic choriomeningitis virus mx strain does not induce the expression of tumor-associated carbonic anhydrase IX in persistently infected HeLa cells.

Summary. - Lymphocytic choriomeningitis virus (LCMV) is an arenavirus that readily causes persistent infections, in which it does not interfere with vital functions of the cells, but can affect expression of "luxurious" genes. MX strain of LCMV (MX LCMV) has been identified as an agent transmissible by cell-to-cell contact in the human carcinoma MaTu cells grown in a mixed culture with HeLa cells. When compared to uninfected HeLa, the MaTu-MX-infected HeLa cells, to which the virus was transmitted via co-cultivation with mitomycin C-treated MaTu cells, showed an elevated expression of a protein called MN, suggesting that MN can be induced by MX LCMV. MN protein was later identified as the carbonic anhydrase isoform IX (CA IX), whose expression has been predominantly associated with hypoxic tumors of poor prognosis. Since the proposal that MX LCMV can induce such a cancer-related protein could substantially change our view on the biology of LCMV-host interaction we undertook its verification. Instead of co-cultivation, we used MaTu cell-free extracts to transmit MX LCMV to HeLa cells. These cells were then grown for more than 30 passages, but the level of MN/CA IX did not increase throughout the whole cultivation period as compared to uninfected HeLa cells. Moreover, a treatment of MaTu-MX-infected HeLa cells with ribavirin eliminated the virus, but did not reduce the MN/CA IX expression. Our results clearly showed that MN/CA IX is independent of MX LCMV and that the virus itself does not influence the MN/CA IX level in HeLa cells.

Expression of hypoxia-inducible carbonic anhydrase-9 relates to angiogenic pathways and independently to poor outcome in non-small cell lung cancer.

Carbonic anhydrase-9 (CA9), a transmembrane enzyme with an extracellular active site, is involved in the reversible metabolism of the carbon dioxide to carbonic acid. Up-regulation of CA by hypoxia and the hypoxia-inducible factor (HIF) pathway has been recently postulated (Wykoff et al. Cancer Res., 60: 7075-7083, 2000). In the present study we examined the expression of this enzyme in non-small cell lung cancer. Of 107 cases analyzed, 39 (36.4%) had strong membrane/cytoplasmic expression of CA9 and were grouped as positive. The staining was confined around areas of necrosis, and a significant association of CA9 expression with the extent of necrosis was noted (P = 0.004). Nevertheless, 38 of 74 cases with focal or extensive necrosis did not express CA9. CA9 expression was more frequent in the squamous cell histology (P = 0.001) and with advanced T stage (P = 0.009). A significant coexpression of CA9 with platelet-derived endothelial cell growth factor and basic fibroblast growth factor receptor expression was noted. Double staining of CA9 with anti-CD31 monoclonal antibody revealed an overall higher microvessel density in the areas expressing CA9 than in negative areas (P = 0.0005). Thirty-one of 38 CA9-positive cases were positive for HIF1a/HIF2a, but HIF positivity was a more common event (68 of 107) and their patterns of expression were diffuse (not confined in the necrotic areas). A direct association of CA9 expression with epidermal growth factor receptor, c-erbB-2, and MUC1 expression was also noted (P < 0.04). Survival analysis showed that CA9 expression is related to poor prognosis. CA9 expression in tumors with low vascularization defined a prognosis similar to the one of patients with highly angiogenic tumors. Multivariate analysis revealed that CA9 expression is a significant prognostic factor independent of angiogenesis. We conclude that CA9 is an important molecule in non-small cell lung cancer, the up-regulation of which occurs in highly hypoxic/necrotic regions of the tumors. The expression of CA9 is linked to the expression of a constellation of proteins involved in angiogenesis, apoptosis inhibition, and cell-cell adhesion disruption, which explains the strong association of CA9 with poor outcome.

Carbonic anhydrase IX, an endogenous hypoxia marker, expression in head and neck squamous cell carcinoma and its relationship to hypoxia, necrosis, and microvessel density.

Carbonic anhydrase IX (CA IX) is a transmembrane glycoprotein with an active extracellular enzyme site. We have shown previously that it was hypoxia inducible and may therefore be an endogenous marker of hypoxia. It is overexpressed in some tumors, particularly renal cell carcinoma. The aim of this study was to examine the expression and localization of CA IX in head and neck squamous cell carcinoma (HNSCC) and relate this to the location of tumor microvessels, angiogenesis, necrosis, and stage. Expression of CA IX was determined by immunoblotting in three HNSCC cell lines grown in normoxia and hypoxia (pO(2) 0.1%) and three paired tumor and normal tissue samples of HNSCC. Archived paraffin sections (79) of HNSCC were immunostained with antibodies to CA IX and CD34 to determine microvessel density (MVD). By double staining sections with CA IX and CD34, the distance between blood vessels and the start of CA IX expression and necrosis was calculated. CA IX was induced by hypoxia in all three HNSCC cell lines and overexpressed in HNSCC tumor tissue. Overexpression was localized to the perinecrotic area of the tumor on immunostaining, and the percentage area of the tumor expressing CA IX was significantly higher with more tumor necrosis (P = 0.001), a high MVD (P = 0.02), and advanced stage (P = 0.033) on univariate analysis and necrosis (P = 0.0003) and MVD (P = 0.0019) on multivariate analysis. The median distance between a blood vessel and the start of CA IX expression was 80 microm (range, 40-140 microm). CA IX is overexpressed in HNSCC because of hypoxia and is a potential biomarker for hypoxia in this tumor. Overexpression may help to maintain the intracellular pH, giving tumor cells a survival advantage and enhancing resistance to radiotherapy and chemotherapy. CA IX is a potential target for future therapy in HNSCC.

Hypoxia-inducible expression of tumor-associated carbonic anhydrases.

The transcriptional complex hypoxia-inducible factor-1 (HIF-1) has emerged as an important mediator of gene expression patterns in tumors, although the range of responding genes is still incompletely defined. Here we show that the tumor-associated carbonic anhydrases (CAs) are tightly regulated by this system. Both CA9 and CA12 were strongly induced by hypoxia in a range of tumor cell lines. In renal carcinoma cells that are defective for the von Hippel-Lindau (VHL) tumor suppressor, up-regulation of these CAs is associated with loss of regulation by hypoxia, consistent with the critical function of pVHL in the regulation of HIF-1. Further studies of CA9 defined a HIF-1-dependent hypoxia response element in the minimal promoter and demonstrated that tight regulation by the HIF/pVHL system was reflected in the pattern of CA IX expression within tumors. Generalized up-regulation of CA IX in VHL-associated renal cell carcinoma contrasted with focal perinecrotic expression in a variety of non-VHL-associated tumors. In comparison with vascular endothelial growth factor mRNA, expression of CA IX demonstrated a similar, although more tightly circumscribed, pattern of expression around regions of necrosis and showed substantial although incomplete overlap with activation of the hypoxia marker pimonidazole. These studies define a new class of HIF-1-responsive gene, the activation of which has implications for the understanding of hypoxic tumor metabolism and which may provide endogenous markers for tumor hypoxia.

Carbonic anhydrase (CA IX) expression, a potential new intrinsic marker of hypoxia: correlations with tumor oxygen measurements and prognosis in locally advanced carcinoma of the cervix.

There is increasing evidence that hypoxia-regulated gene expression influences tumor aggressiveness, contributing to the poorer outcome of patients with hypoxic tumors. The role of the transcriptional complex hypoxia-inducible factor-1 as an important mediator of hypoxia-regulated gene expression is one of the best documented pathways. Recently, it has emerged that certain tumor-associated carbonic anhydrases (CAs) can be added to the list of known hypoxia-inducible factor-responsive genes. Here we show that the immunohistochemical expression of the tumor-associated CA IX is correlated with the level of hypoxia in human cervical tumors. We performed a prospective study in 68 patients where needle electrodes were used to make direct measurements of tumor oxygenation levels. CA IX expression was evaluated immunohistochemically in pretreatment tumor biopsies. There was a significant positive correlation between the level of tumor hypoxia (HP5) and the extent of CA IX expression. A retrospective study of 130 squamous cell cervical carcinomas demonstrated that a semiquantitative immunohistochemical analysis of CA IX expression in tumor biopsies is a significant and independent prognostic indicator of overall survival and metastasis-free survival after radiation therapy. These studies provide clinical evidence that CA IX expression is up-regulated in hypoxic human cervical tumors and is associated with a poor prognosis. CA IX may act as an intrinsic marker of tumor hypoxia and poor outcome after radiation therapy. The level of CA IX expression may be used to aid in the selection of patients who would benefit most from hypoxia-modification therapies or bio-reductive drugs.

Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment.

MN is a transmembrane glycoprotein that has been detected in HeLa cells and in some human carcinomas. The expression of MN protein in HeLa cells is regulated by cell density. In HeLa x fibroblast cell hybrids its expression correlates with tumorigenicity. Using a specific monoclonal antibody we have identified a cDNA clone coding for MN. Analysis of the deduced amino acid sequence revealed strong structural homology between the central region of the MN protein and carbonic anhydrases (CA). MN sequence retains the conserved zinc-binding site as well as the enzyme's active center. In accord with these findings, MN protein from HeLa cells was found to bind zinc and to have carbonic anhydrase activity. The N-terminal region of MN shares some similarity with DNA binding proteins of the helix-loop-helix (HLH) family, and the protein was found to have affinity for DNA by DNA-cellulose chromatography. The region between the CA-like domain and the putative HLH domain is rich in imperfect repeats of serine, proline, glycine and acidic residues with few hydrophobic amino acids, resembling thus an activation region of transcription factors. The fact that MN protein is detectable in several types of human carcinomas, but not in corresponding non-cancerous tissues, suggests its possible role in neoplasia. In addition, the analysis of biological consequences of MN expression of NIH3T3 cells provides the evidence in favour of MN protein involvement in control of cell proliferation and transformation.

P53 tumour suppressor modulates transcription of the TATA-less gene coding for the tumour-associated carbonic anhydrase MN/CA IX in MaTu cells.

MN/CA IX (MN) exhibits a strong association with tumours. Co-transfection experiments revealed that in MaTu cells the activity of the (-173;+31) MN promoter construct was repressed by the wild type p53 in a dose-responsive manner and stimulated by the (143(Val-->Ala)) mutant. Upregulation of endogenous p53 by mitomycin C treatment in MaTu cells also had a profound effect on MN expression as well as the activity of MN promoter in a reporter construct. p53 can thus modulate MN expression and at least in a subset of tumours the changed p53 status might be responsible for MN positivity. Co-transfections with internally deleted MN promoter constructs demonstrated that the wild type p53 exerts its repression activity on the level of the basal transcriptional machinery and not on a particular cis element within the MN promoter.

Prognostic significance of a novel hypoxia-regulated marker, carbonic anhydrase IX, in invasive breast carcinoma.

PURPOSE: To assess the frequency of expression and the prognostic significance of a hypoxia-regulated marker, carbonic anhydrase IX (CA IX), in a cohort of patients with invasive breast cancer. PATIENTS AND METHODS: CA IX expression was evaluated by immunohistochemistry with a murine monoclonal antibody, M75, in a series of 103 women treated surgically for invasive breast cancer. The majority of patients were treated with adjuvant hormonal or chemotherapy. The frequency of CA IX expression, its association with recognized prognostic factors, and the relationship with outcome was evaluated by univariate and multivariate statistical analyses. RESULTS: CA IX expression was present in 49 (48%) of 103 cases. The level of CA IX expression was found to be significantly associated with tumor necrosis (P <.001), higher grade (P =.02), and negative estrogen receptor status (P <.001). Furthermore, CA IX expression was associated with a higher relapse rate (P =.004) and a worse overall survival (P =.001). By multivariate analysis, CA IX was also shown to be an independent predictive factor for overall survival (hazard ratio, 2.61; 95% confidence interval, 1.01 to 6.75, P =.05). CONCLUSION: CA IX expression was associated with worse relapse-free survival and overall survival in an unselected cohort of patients with invasive breast carcinoma. The potential role of CA IX as a marker of hypoxia within breast carcinomas was also indicated by a significant association with necrosis. Further work assessing its prognostic significance in breast cancer is warranted, particularly interactions with radiotherapy and chemotherapy resistance.

Hypoxia-regulated carbonic anhydrase-9 (CA9) relates to poor vascularization and resistance of squamous cell head and neck cancer to chemoradiotherapy.

PURPOSE: Carbonic anhydrases are proteins involved in the catalytic hydration of carbon dioxide to carbonic acid. Recent studies show that carbonic anhydrase 9 (CA9) is up-regulated by hypoxia and that its immunohistochemical tissue distribution follows the distribution of the radiosensitizer pimonidazole (C. C. Wykoff et al., Cancer Res. 60: 7075-7083, 2001). Therefore, CA9 expression may show hypoxia levels of clinical importance. EXPERIMENTAL DESIGN: We assessed the expression of CA9 and the microvessel density (MVD; CD31-positive) in 75 locally advanced squamous cell head and neck cancers treated with concurrent chemoradiotherapy with carboplatin. RESULTS: Strong membrane/cytoplasmic CA9 expression, noted in 20/75 (26.6%) tumors, mainly occurred in tumors with very poor vascularization (expression in 63% versus 14%; P < 0.0001), was located around areas of focal necrosis, and was related to poor complete response rate (40% versus 70%; P = 0.02). These observations suggested that CA9 might be a marker of clinically important hypoxia. Combining the CA9 staining and the tumor angiogenicity (MVD), we identified three groups of patients: (a) hypoxic tumors; (b) euoxic highly angiogenic tumors; and (c) euoxic non-highly angiogenic tumors. Groups (a) and (b) had a very poor local relapse-free survival (P < 0.0001). CONCLUSIONS: Stratification of patients undergoing radical radiotherapy using the CA9/MVD model may be useful for the individualization of therapeutic strategies combining antiangiogenesis and hypoxia targeting with radiotherapy.

Expression of S100P gene in cervical carcinoma cells is independent of E7 human papillomavirus oncogene.

High-risk human papillomaviruses (HPV) significantly contribute to development of cervical cancer. HPV E7 oncoprotein interferes with the control of cell growth via functional inactivation and/or regulation of multiple molecular targets. Induction of ectopic E7 in breast carcinoma cells has been proposed to decrease transcription of S100P gene, which encodes a calcium-binding protein associated with different types of tumors. We examined a possible relationship between E7 and S100P genes in cervical cell lines. RT-PCR analysis revealed that all HPV-positive cell lines expressed approximately equal levels of E7. Out of them, HeLa, CGL3 and SiHa carcinoma cells as well as HCE16/3 immortalized cells expressed also S100P gene. Inhibition of a DNA methylation by 5-aza-2'-deoxycytidine (5-aza-dC) in S100P-negative cell lines CGL1 and Caski resulted in induced transcription of S100P, but the normal S100P level in SiHa cells was not further increased. Our results suggest that S100P gene expression is independent of E7 in cervical cell lines and that at least in some cases it is subjected to regulation by DNA methylation.

Aztreonam plus clindamycin as therapy for pelvic infections in women.

Aztreonam, a new monobactam antibiotic with specific gram-negative aerobic activity, was used in combination with clindamycin in the treatment of 40 women with pelvic infection, including post-partum endometritis, pelvic inflammatory disease, and post-hysterectomy pelvic cellulitis. Clinical cure was achieved in 87 percent of patients. Failure was related to the limited gram-positive aerobic spectrum of clindamycin. All aerobic gram-negative enteric organisms were sensitive in vitro to less than 0.125 microgram/ml of aztreonam.

Comparison of ticarcillin plus clavulanic acid with cefoxitin in the treatment of female pelvic infection.

Ninety-three female patients with post-cesarean endometritis, post-hysterectomy pelvic cellulitis, and other miscellaneous moderately severe pelvic soft-tissue infections were treated in a randomized fashion with either ticarcillin plus clavulanic acid or cefoxitin. Of the 47 patients treated with ticarcillin plus clavulanic acid, 38 had clinical cures, four showed improvement, therapy failed in three, and two were nonevaluable, for a failure rate of 6.7 percent. Of the 46 patients treated with cefoxitin, 33 had clinical cures, five showed improvement, therapy failed in seven, and one was nonevaluable, for a failure rate of 15.6 percent. Bacteriologically, the addition of clavulanic acid to ticarcillin was found to broaden the antibacterial spectrum to include some Escherichia coli, most Klebsiella, many coagulase-negative staphylococci, and all isolates of Staphylococcus aureus. Adverse reactions were few, with only one patient having therapy with cefoxitin discontinued because of side effects. It is concluded that ticarcillin plus clavulanic acid is quite suitable for antibiotic therapy of female pelvic soft-tissue infection, based on the (expanded) coverage of both aerobic and anaerobic bacterial species.

Immunohistochemistry of carbonic anhydrase isozyme IX (MN/CA IX) in human gut reveals polarized expression in the epithelial cells with the highest proliferative capacity.

MN/CA IX is a recently discovered member of the carbonic anhydrase (CA) gene family that has been identified in the plasma membranes of certain tumor and epithelial cells and found to promote cell proliferation when transfected into NIH3T3 cells. This study presents localization of MN/CA IX in human gut and compares its distribution to those of CA I, II, and IV, which are known to be expressed in the intestinal epithelium. The specificity of the monoclonal antibody for MN/CA IX was confirmed by Western blots and immunostaining of COS-7 cells transfected with MN/CA IX cDNA. Immunohistochemical stainings of human gut revealed prominent polarized staining for MN/CA IX in the basolateral surfaces of the enterocytes of duodenum and jejunum, the reaction being most intense in the crypts. A moderate reaction was also seen in the crypts of ileal mucosa, whereas the staining became generally weaker in the large intestine. The results indicate isozyme-specific regulation of MN/CA IX expression along the cranial-caudal axis of the human gut and place the protein at the sites of rapid cell proliferation. The unique localization of MN/CA IX on the basolateral surfaces of proliferating crypt enterocytes suggests that it might serve as a ligand or a receptor for another protein that regulates intercellular communication or cell proliferation. Furthermore, MN/CA IX has a completely conserved active site domain of CAs suggesting that it could also participate in carbon dioxide/bicarbonate homeostasis.

Transient transformation of mammalian cells by MN protein, a tumor-associated cell adhesion molecule with carbonic anhydrase activity.

The MN protein is associated with certain human carcinomas, but absent in most normal tissues. It is a transmembrane protein; its extracellular part contains a domain homologous with carbonic anhydrases (CAs) and a proteoglycan-like region. In the present study, we observed that cells (human CGL1 and mouse NIH3T3 cells) transfected with MN cDNA showed morphologic transformation, but reverted to normal phenotype after 4-5 weeks. This reversion was not due to the loss, silencing, or mutations of MN insert. We also found that MN protein exerted CA enzymatic activity, but this was not relevant for morphologic transformation of cells. MN is an adhesion protein, involved in cell-to-cell contacts, this probably could explain its role in tumorigenesis.

Up-regulation of p53 by antisense expression of HPV18 E6 oncogene does not influence the level of MN/CA IX tumor-associated protein in HeLa cervical carcinoma cells.

Oncogenic potential of human papillomaviruses is related to capacity of HPV-encoded oncoproteins to bind and inactivate tumor suppressor proteins. Interaction of p53 with HPV E6 results in aberrant regulation of various cellular genes. We evaluated the possible involvement of MN/CA9 gene, whose expression is closely associated with cervical carcinomas, in regulatory pathways driven by p53 and E6. We demonstrated that one of the two p53 consensus sequences present in MN/CA9 promoter participates in DNA-protein interaction but it does not bind p53. Tetracycline-inducible antisense expression of HPV18 E6 in human cervical carcinoma HeLa cells resulted in increased level of p53 but did not affect expression of MN/CA IX protein. Therefore we conclude that at least in HeLa cells there is no direct relationship between expression of MN/CA IX and expression of E6 or p53.

Fine-tuning the fluorescent antibody test for chlamydial infections in pregnancy.

Endocervical specimens from 168 asymptomatic women in the second trimester of pregnancy were screened for Chlamydia trachomatis infection with both standard tissue culture methods and a direct fluorescein-conjugated monoclonal antibody assay (MicroTrak). Based on tissue culture results, the prevalence of Chlamydia trachomatis was 26.2% in this population. Compared with tissue culture, the monoclonal antibody assay's sensitivity, specificity, and positive predictive value varied depending upon how many elementary bodies were used to define a positive test. Specifically, if a cutoff of ten elementary bodies was used (as per the manufacturer's instructions), the sensitivity, specificity, and positive predictive value of this test were 86.3, 98.4, and 95.0%, respectively. At the other extreme, a cutoff of one elementary body produced a more sensitive but less specific test, with parameters of 93.2, 89.5, and 75.9%, respectively. Based on these data and operating characteristic analysis, the cutoff value defining a positive test was appropriately set at two or more elementary bodies, at least for this study population. This resulted in a sensitivity, specificity, and positive predictive value of 93.2, 95.2, and 87.2%, respectively. This monoclonal antibody assay appears to be a reasonable substitute for cell culture for Chlamydia trachomatis. However, because of varying magnitudes and implications of false-positive and false-negative tests, clinicians are urged to determine the appropriate breakpoint for their individual laboratories and patient populations before substituting the direct fluorescent antibody test for tissue culture.