Infrared spectroscopic laser scanning confocal microscopy for whole-slide chemical imaging.

Chemical imaging, especially mid-infrared spectroscopic microscopy, enables label-free biomedical analyses while achieving expansive molecular sensitivity. However, its slow speed and poor image quality impede widespread adoption. We present a microscope that provides high-throughput recording, low noise, and high spatial resolution where the bottom-up design of its optical train facilitates dual-axis galvo laser scanning of a diffraction-limited focal point over large areas using custom, compound, infinity-corrected refractive objectives. We demonstrate whole-slide, speckle-free imaging in ~3 min per discrete wavelength at 10× magnification (2 µm/pixel) and high-resolution capability with its 20× counterpart (1 µm/pixel), both offering spatial quality at theoretical limits while maintaining high signal-to-noise ratios (>100:1). The data quality enables applications of modern machine learning and capabilities not previously feasible - 3D reconstructions using serial sections, comprehensive assessments of whole model organisms, and histological assessments of disease in time comparable to clinical workflows. Distinct from conventional approaches that focus on morphological investigations or immunostaining techniques, this development makes label-free imaging of minimally processed tissue practical.

Colon Cancer Grading Using Infrared Spectroscopic Imaging-Based Deep Learning.

Tumor grade assessment is critical to the treatment of cancers. A pathologist typically evaluates grade by examining morphologic organization in tissue using hematoxylin and eosin (H&E) stained tissue sections. Fourier transform infrared spectroscopic (FT-IR) imaging provides an alternate view of tissue in which spatially specific molecular information from unstained tissue can be utilized. Here, we examine the potential of IR imaging for grading colon cancer in biopsy samples. We used a 148-patient cohort to develop a deep learning classifier to estimate the tumor grade using IR absorption. We demonstrate that FT-IR imaging can be a viable tool to determine colorectal cancer grades, which we validated on an independent cohort of surgical resections. This work demonstrates that harnessing molecular information from FT-IR imaging and coupling it with morphometry is a potential path to develop clinically relevant grade prediction models.

INFORM: INFrared-based ORganizational Measurements of tumor and its microenvironment to predict patient survival.

The structure and organization of a tumor and its microenvironment are often associated with cancer outcomes due to spatially varying molecular composition and signaling. A persistent challenge is to use this physical and chemical spatial organization to understand cancer progression. Here, we present a high-definition infrared imaging-based organizational measurement framework (INFORM) that leverages intrinsic chemical contrast of tissue to label unique components of the tumor and its microenvironment. Using objective and automated computational methods, further, we determine organization characteristics important for prediction. We show that the tumor spatial organization assessed with this framework is predictive of overall survival in colon cancer that adds to capability from clinical variables such as stage and grade, approximately doubling the risk of death in high-risk individuals. Our results open an all-digital avenue for measuring and studying the association between tumor spatial organization and disease progression.

Safety and Efficacy of Apixaban vs Enoxaparin for Preventing Postoperative Venous Thromboembolism in Women Undergoing Surgery for Gynecologic Malignant Neoplasm: A Randomized Clinical Trial.

Importance: Current guidelines recommend a 28-day course of enoxaparin for thromboprophylaxis after surgery for gynecologic cancer. The high cost of this medication and the low adherence rates observed in prior studies provide an opportunity to benefit patients by demonstrating the safety of a more cost-effective, easier to use thromboprophylactic. Objective: To investigate the safety and efficacy of an oral treatment alternative for thromboprophylaxis in postoperative patients with gynecologic cancer. Design, Setting, and Participants: This was a patient-based, multicenter, open-label, blinded, end point, randomized clinical trial conducted May 2015 to March 2019 in outpatient and inpatient gynecologic oncology settings. Women undergoing surgery for suspected or confirmed gynecologic cancer were approached for recruitment. The trial compared rates of major bleeding and clinically relevant nonmajor bleeding events during a 90-day follow-up period in patients taking apixaban or enoxaparin for postoperative thromboprophylaxis using a modified intent-to-treat analysis. Data analysis was performed from October to December 2019. Interventions: Women were randomized to 28 days of apixaban (2.5 mg orally twice daily) or enoxaparin (40 mg subcutaneously daily). Main Outcomes and Measures: The primary outcome was major bleeding and clinically relevant nonmajor bleeding events. Secondary outcomes included incidence of venous thromboembolic events, adverse events, medication adherence, participant quality of life, and medication satisfaction. Results: Of 500 women recruited for the study, 400 were enrolled and randomized (median age, 58.0 years; range, 18.0-89.0 years); 204 received apixaban and 196 received enoxaparin. Treatment groups did not differ in terms of race/ethnicity, cancer stage, or surgery modality (open vs robotic). There were no statistically significant differences between the apixaban and enoxaparin groups in terms of rates of major bleeding events (1 patient [0.5%] vs 1 patient [0.5%]; odds ratio [OR], 1.04; 95% CI, 0.07-16.76; P > .99), clinically relevant nonmajor bleeding events (12 patients [5.4%] vs 19 patients [9.7%]; OR, 1.88; 95% CI, 0.87-4.1; P = .11), venous thromboembolic events (2 patients [1.0%] vs 3 patients [1.5%]; OR, 1.57; 95% CI, 0.26-9.50; P = .68), adverse events, medication adherence, or quality of life between the groups. Participant satisfaction was significantly greater in the apixaban group with regard to ease of taking the medication (186 patients [98.9%] vs 110 patients [58.8%]; OR, 0.06; 95% CI, 0.01-0.25; P < .001) and pain associated with taking the medication (4 patients [2.1%] vs 92 patients [49.2%]; OR, 9.20; 95% CI, 2.67-31.82; P < .001). Conclusions and Relevance: These findings suggest that oral apixaban is a potentially safe, less painful, and easier-to-take alternative to subcutaneous enoxaparin for thromboprophylaxis after surgery for gynecologic cancer. The efficacy of apixaban to prevent venous thromboembolic events is hypothesized as being equivalent. Trial Registration: ClinicalTrials.gov Identifier: NCT02366871.

Histopathology Discrepancy of Preoperative Endometrial Sampling and Final Specimen: How Does This Influence Selective Lymph Node Dissection?

OBJECTIVE: Preoperative histology is a major component in the perioperative selective lymph node (LN) dissection decision process. Discrepancy between preoperative endometrial sampling and final specimen histopathology is generally accepted. The goals of this project are to determine if discrepancy of histopathology is associated with alteration of adjuvant treatment or outcome. MATERIALS AND METHODS: We performed a retrospective cross-sectional analysis of all patients undergoing surgery for endometrial cancer at a single institution from 2010 to 2014. All patients had preoperative endometrial sampling. Histopathology discrepancy was evaluated for potential in variation of perioperative LN dissection. Criteria for not performing LN dissection was defined as preoperative endometrioid histology, grade 1 or 2 lesion, myometrial invasion of 50% or less, and primary tumor diameter 2 cm or less. RESULTS: A total of 352 patients were identified; 44 were excluded because of no preoperative pathology or no residual disease on final pathology. Discrepancy of histopathology was noted in 64/308 (20.8%; 95% confidence interval [CI], 16.2%-25.3%) patients. Preoperative endometrioid histology was noted in 272 patients, and 17/272 (6.3%; 95% CI, 3.4%-9.1%) had preoperative sampling reviewed as a grade 1 or 2 endometrioid lesion and final specimen was upgraded to grade 3. Downstaging occurred in 3/272 (1.1%; 95% CI, 0%-2.3%) patients with preoperative grade 3 lesion and final specimen demonstrated grade 1 or 2 disease. All 3 patients' primary tumor diameter was greater than 2 cm and therefore received LN dissection. Histopathological discrepancy that would alter perioperative LN dissection decision based on the aforementioned criteria occurred in 2/272 (0.7%; 95% CI, 0%-1.8%). CONCLUSIONS: Despite a 20% discrepancy of preoperative and postoperative histopathology, discrepancy that would alter a perioperative decision for LN dissection occurs in only 0.7% of cases in this retrospective single-institutional experience. Myometrial invasion and tumor size may be more influential than histology in LN selection criteria.

Soluble guanylate cyclase as an alternative target for bronchodilator therapy in asthma.

Asthma is defined by airway inflammation and hyperresponsiveness, and contributes to morbidity and mortality worldwide. Although bronchodilation is a cornerstone of treatment, current bronchodilators become ineffective with worsening asthma severity. We investigated an alternative pathway that involves activating the airway smooth muscle enzyme, soluble guanylate cyclase (sGC). Activating sGC by its natural stimulant nitric oxide (NO), or by pharmacologic sGC agonists BAY 41-2272 and BAY 60-2770, triggered bronchodilation in normal human lung slices and in mouse airways. Both BAY 41-2272 and BAY 60-2770 reversed airway hyperresponsiveness in mice with allergic asthma and restored normal lung function. The sGC from mouse asthmatic lungs displayed three hallmarks of oxidative damage that render it NO-insensitive, and identical changes to sGC occurred in human lung slices or in human airway smooth muscle cells when given chronic NO exposure to mimic the high NO in asthmatic lung. Our findings show how allergic inflammation in asthma may impede NO-based bronchodilation, and reveal that pharmacologic sGC agonists can achieve bronchodilation despite this loss.

Cotyledonoid dissecting leiomyoma with adipocytic differentiation: A case report.

•The second case of cotyledonoid dissecting lipoleiomyoma documented in the literature is reported.•Cotyledonoid dissecting leiomyoma presents in a similar manner as aggressive malignancies; however, it is a benign lesion.•Recurrence of cotyledonoid dissecting leiomyoma is exceedingly rare with only one documented recurrence following conservative treatment.

Lymphovascular space invasion in robotic surgery for endometrial cancer.

BACKGROUND: Minimally invasive surgery has become a standard treatment for endometrial cancer and offers significant benefits over abdominal approaches. There are discrepant data regarding lymphovascular space invasion (LVSI) and positive peritoneal cytology with the use of a uterine manipulator, with previous small-scale studies demonstrating an increased incidence of these prognostically important events. We sought to determine if there was a higher incidence of LVSI in patients who underwent robot-assisted surgery for endometrial cancer. METHODS: We performed a single-institution review of medical records for patients who underwent open abdominal or robot-assisted hysterectomy for endometrial cancer over a 24-month period. The following data were abstracted: age, tumor grade and stage, size, depth of invasion, LVSI, and peritoneal cytology. For patients with LVSI, slides were reviewed by 2 pathologists for confirmation of LVSI. RESULTS: Of 104 patients identified, LVSI was reported in 39 (37.5%) and positive peritoneal cytology in 6 (4.8%). Rates of peritoneal cytology were not significantly different between the 2 groups (odds ratio, 0.55; 95% confidence interval, 0.10-3.17; P=.50). LVSI was reported in significantly fewer robot-assisted hysterectomies than open procedures (odds ratio, 0.39; 95% confidence interval, 0.17-0.92; P=.03). In subgroup analyses restricted to early-stage disease (stage≤II), there was no significant difference in LVSI between open and robot-assisted hysterectomies (odds ratio, 0.64; 95% confidence interval, 0.22-1.85; P=.43). CONCLUSION: In this retrospective study, we found that use of a uterine manipulator in robot-assisted surgery did not increase the incidence of LVSI.

Outcomes of women with atypical glandular cells on preoperative cytology and endometrial cancer.

OBJECTIVE: This study aims to examine the prognostic importance of preoperative cervical cytologic diagnosis with atypical glandular cells (AGC) or malignant cells (MC) as a predictor of poor outcomes in endometrial cancer. MATERIALS AND METHODS: A total of 563 patients were surgically staged for endometrial adenocarcinoma from 2002 to 2012 at our institution. Of these patients, 106 were included to perform a case-control study (39 patients with AGC or MC and 67 controls). Included patients were not significantly different from excluded patients and were matched for age, race, and body mass index. Outcome variables included presence of extrauterine disease (International Federation of Gynecology and Obstetrics stage ≥II) and high intermediate risk (HIR) disease. Further analysis sought to improve the prediction combining AGC or MC with other factors, such as grade and CA-125 levels. Standard statistical analyses were used. RESULTS: Among the patients with AGC or MC, 53.8% had HIR disease compared with 30.3% with normal cervical cytologic diagnosis (odds ratio [OR], 2.68; 95% confidence interval [CI], 1.18-6.09; P = 0.02). Extrauterine disease was found in 43.6% of patients with AGC or MC compared with that of 15.2% in patients with normal cervical cytologic diagnosis (OR, 4.33; 95% CI, 1.72-10.90; P < 0.01). Multivariate analysis confirmed that AGC or MC was an independent predictor of HIR disease (OR, 8.41; 95% CI, 1.34-52.78; P = 0.02) and extrauterine disease (OR, 4.78; 95% CI, 1.26-18.1; P = 0.02). The combination of elevated CA-125 levels with AGC or MC cervical cytologic diagnosis increased the statistical prediction of extrauterine disease (OR, 13.3; 95% CI, 3.1-56.8; P < 0.01) and HIR disease (OR, 5.83; 95% CI, 1.44-23.71; P = 0.02). CONCLUSIONS: Patients with AGC or MC on preoperative cervical cytology are at risk for extrauterine and HIR disease. These preoperative findings should warn surgeons of the potential of extrauterine or occult metastatic disease.

Temporal analysis of Candida albicans gene expression during biofilm development.

Microarrays were used to identify changes in gene expression associated with Candida albicans biofilm development. Two biofilm substrates (denture and catheter), and two C. albicans strains for each substrate, were tested to remove model- and strain-dependent variability from the overall dataset. Three biofilm developmental phases were examined: early (6 h), intermediate (12 h), and mature (48 h). Planktonic specimens were collected at the same time points. Data analysis focused primarily on gene expression changes over the time-course of biofilm development. Glycolytic and non-glycolytic carbohydrate assimilation, amino acid metabolism, and intracellular transport mechanisms were important during the early phase of biofilm formation. These early events increase intracellular pools of pyruvate, pentoses and amino acids, which prepare the biofilm for the large biomass increase that begins around 12 h of development. This developmental stage demands energy and utilizes specific transporters for amino acids, sugars, ions, oligopeptides and lactate/pyruvate. At mature phase (48 h), few genes were differentially expressed compared with the 12 h time point, suggesting a relative lack of initiation of new metabolic activity. Data analysis to assess biofilm model-specific gene expression showed more dynamic changes in the denture model than in the catheter model. Data analysis to identify gene expression changes that are associated with each strain/substrate combination identified the same types of genes that were identified in the analysis of the entire dataset. Collectively, these data suggest that genes belonging to different, but interconnected, functional categories regulate the morphology and phenotype of C. albicans biofilm.

RT-PCR analysis of Candida albicans ALS gene expression in a hyposalivatory rat model of oral candidiasis and in HIV-positive human patients.

ALS gene expression was studied in the hyposalivatory rat model of oral candidiasis and in clinical specimens collected from HIV-positive patients to assess similarities in expression patterns between the model system and clinical isolates. Two Candida albicans strains, SC5314 and OY-2-76, were used in the rat model system and infection progressed for 3 or 5 days. The strains produced similar oral lesions at 3 days. At 5 days, strain OY-2-76 produced more superficial lesions containing relatively more yeast forms compared to invasive hyphal forms observed for strain SC5314. For all infections, the most severe lesions were observed on the tongue and gingiva overlying the mandible. ALS transcripts were easier to detect by RT-PCR later in infection and under other conditions where more fungal cells were present. Expression of ALS1, ALS2, ALS3 and ALS4 was observed in rats infected for 3 days with ALS5 and ALS9 transcripts detected after 5 days of infection. Expression of ALS6 was observed in a single specimen from a 5-day infection while ALS7 transcript was never found. Expression of all ALS genes was observed in oral clinical material collected from HIV-positive patients although ALS6 and ALS7 transcripts required an extra PCR amplification step to be detected. Overall, the patterns of ALS gene expression were similar between the rat model and human clinical specimens, suggesting that the model would be useful for studying the phenotype of al delta/al delta mutant strains.

Cellular and molecular biology of Candida albicans estrogen response.

Candida albicans is the most common etiological agent of vaginal candidiasis. Elevated host estrogen levels and the incidence of vaginal candidiasis are positively associated. Elevated estrogen levels may affect host and/or fungal cells. This study investigates the effect of 17-beta-estradiol, 17-alpha-estradiol, ethynyl estradiol, and estriol on several C. albicans strains at concentrations ranging from 10(-5) to 10(-10) M. The addition of 17-beta-estradiol or ethynyl estradiol to C. albicans cells caused an increase in the number of cells forming germ tubes and an increase in germ tube length in a dose- and strain-dependent manner. The addition of 17-alpha-estradiol or estriol did not have a significant effect on germ tube formation by the cultured cells. Exposure to exogenous estrogens did not significantly change the biomass of any C. albicans culture tested. The transcriptional profile of estrogen-treated C. albicans cells showed increased expression of CDR1 and CDR2 across several strain-estrogen concentration-time point combinations, suggesting that these genes are the most responsive to estrogen exposure. Analysis of strain DSY654, which lacks the CDR1 and CDR2 coding sequences, showed a significantly decreased number of germ tube-forming cells in the presence of 17-beta-estradiol. PDR16 was the most highly up-regulated gene in strain DSY654 under these growth conditions. The cell biology and gene expression data from this study led to a model that proposes how components of the phospholipid and sterol metabolic pathways may interact to affect C. albicans germ tube formation and length.

Functional specificity of Candida albicans Als3p proteins and clade specificity of ALS3 alleles discriminated by the number of copies of the tandem repeat sequence in the central domain.

Candida albicans strain SC5314 contains two ALS3 alleles, which differ in sequence with respect to the number of copies of the 108 bp tandem repeat sequence within the central domain of the coding region. One allele (ALS3(12)) has 12 tandem repeat copies while the other (ALS3(9)) has 9 copies. Wild-type C. albicans (ALS3(12)/ALS3(9)) and those containing various ALS3 alleles (ALS3(12)/als3Delta(9), als3Delta(12)/ALS3(9) and als3Delta(12)/als3Delta(9)) were assayed for adhesion to monolayers of cultured vascular endothelial and pharyngeal epithelial cells. These assays showed obvious adhesive function for the larger Als3p protein, compared to a minor contribution to adhesion from the smaller protein. These functional differences in strain SC5314 prompted examination of ALS3 allelic diversity across the five major genetic clades of C. albicans. This analysis focused on the number of copies of the tandem repeat sequence within the central domain of the coding region and showed a range of alleles encoding from 6 to 19 tandem repeat copies. Clades differed with respect to prevalent ALS3 alleles and allele distribution, but were similar for the mean number of tandem repeat copies per ALS3 allele. Analysis of allelic pairing showed clade differences and the tendency for C. albicans strains to encode one longer and one shorter ALS3 allele. The allelic variability observed for ALS3 and its functional consequences observed in strain SC5314 highlight the importance of understanding ALS allelic diversity in order to draw accurate conclusions about Als protein function.

Comparison between Candida albicans agglutinin-like sequence gene expression patterns in human clinical specimens and models of vaginal candidiasis.

Expression of the eight genes in the Candida albicans agglutinin-like sequence (ALS) family was studied by reverse transcription-PCR of RNA isolated from clinical vaginal fluid specimens and vaginal candidiasis model systems. Although expression of all ALS genes was detected across the set of clinical specimens, ALS1, ALS2, ALS3, and ALS9 transcripts were detected most frequently, and expression of ALS4 and ALS5 was detected least frequently. Laboratory strain 3153A and two C. albicans strains isolated from the clinical specimens were studied using two models of vaginal candidiasis to determine how closely these models mimicked the clinical specimens at the level of gene expression. ALS gene expression patterns in a murine vaginitis model were identical to those from the clinical specimens. Expression of more ALS genes was detected in specimens collected 7 days after infection compared to those collected at 4 days. Similar patterns of ALS gene expression were observed when the three C. albicans strains were tested in the reconstituted human vaginal epithelium model. In this model, expression of ALS4, ALS5, ALS6, and ALS7 was least frequently detected. Negative or weakened signals for ALS4 expression were observed at early time points, suggesting that ALS4 expression, which was strong in the inoculum cells, was down-regulated upon contact of C. albicans with vaginal epithelial cells in this model. The data presented here support the conclusion of host-site-specific influences on ALS gene expression and validate the use of the experimental models for evaluating the phenotype of als/als mutant strains.

ALS3 and ALS8 represent a single locus that encodes a Candida albicans adhesin; functional comparisons between Als3p and Als1p.

The ALS (agglutinin-like sequence) gene family of Candida albicans encodes eight cell-surface glycoproteins, some of which are involved in adherence to host surfaces. A mutational analysis of each ALS gene is currently being performed to deduce the functions of the encoded proteins and to better understand the role of these proteins in C. albicans biology and pathogenesis. This paper describes construction of an als3/als3 mutant and comparison of its phenotype to an als1/als1 strain. Efforts to disrupt ALS3 indicated that the gene could be deleted in two transformation steps, suggesting that the gene is encoded by a single locus and that the ALS3-like locus, ALS8, does not exist. Strains lacking ALS3 or ALS1 did not exhibit a defect in germ tube formation when grown in RPMI 1640 medium, but the als1/als1 mutant formed significantly fewer germ tubes in Lee medium. Analysis of ALS3 and ALS1 promoter activity using green fluorescent protein (GFP) reporter strains and flow cytometry showed that when cells are placed into medium that promotes germ tube formation, ALS1 is transcribed prior to ALS3. Comparison of the mutant strains in adhesion assays showed that the als3/als3 strain was defective in adhesion to both human umbilical vein endothelial cells (HUVEC) and buccal epithelial cells (BEC), but not to fibronectin-coated plastic plates. In contrast, the als1/als1 strain showed decreased adherence to HUVEC, but adherence to BEC and fibronectin were the same as wild-type controls. Inoculation of the buccal reconstituted human epithelium (RHE) model of oral candidiasis with the mutant strains showed nearly a total lack of adhesion and epithelial destruction by the als3/als3 mutant while the als1/als1 strain showed only a slightly reduced degree of epithelial destruction compared to the wild-type control. Adhesion data presented here suggest that, in the assays performed, loss of Als3p affects C. albicans adhesion more than loss of Als1p. Collectively, these results demonstrate functional similarities and differences between Als1p and Als3p, and suggest the potential for more complex interrelationships between the ALS genes and their encoded proteins.

RT-PCR detection of Candida albicans ALS gene expression in the reconstituted human epithelium (RHE) model of oral candidiasis and in model biofilms.

An RT-PCR assay was developed to analyse expression patterns of genes in the Candida albicans ALS (agglutinin-like sequence) family. Inoculation of a reconstituted human buccal epithelium (RHE) model of mucocutaneous candidiasis with strain SC5314 showed destruction of the epithelial layer by C. albicans and also formation of an upper fungal layer that had characteristics similar to a biofilm. RT-PCR analysis of total RNA samples extracted from C. albicans-inoculated buccal RHE showed that ALS1, ALS2, ALS3, ALS4, ALS5 and ALS9 were consistently detected over time as destruction of the RHE progressed. Detection of transcripts from ALS7, and particularly from ALS6, was more sporadic, but not associated with a strictly temporal pattern. The expression pattern of ALS genes in C. albicans cultures used to inoculate the RHE was similar to that observed in the RHE model, suggesting that contact of C. albicans with buccal RHE does little to alter ALS gene expression. RT-PCR analysis of RNA samples extracted from model denture and catheter biofilms showed similar gene expression patterns to the buccal RHE specimens. Results from the RT-PCR analysis of biofilm RNA specimens were consistent between various C. albicans strains during biofilm development and were comparable to gene expression patterns in planktonic cells. The RT-PCR assay described here will be useful for analysis of human clinical specimens and samples from other disease models. The method will provide further insight into the role of ALS genes and their encoded proteins in the diverse interactions between C. albicans and its host.

The use of synthetic genes for the expression of ciliate proteins in heterologous systems.

The common fish parasite, Ichthyophthirius multifiliis, expresses abundant glycosylated phosphatidylinositol (GPI)-anchored membrane proteins known as immobilization antigens, or i-antigens. These proteins are targets of the host immune response, and have been identified as potential candidates for recombinant subunit vaccine development. Nevertheless, because Ichthyophthirius utilizes a non-standard genetic code, expression of the corresponding gene products, either as subunit antigens in conventional protein expression systems, or as vector-encoded antigens in the case of DNA vaccines, is far from straightforward. To overcome this problem, we utilized 'assembly polymerase chain reaction' to manufacture synthetic versions of two genes (designated IAG52A[G5/CC] and IAG52B[G5/CC]) encoding approximately 52/55 kDa i-antigens from parasite strain G5. This approach made it possible to eliminate unwanted stop codons and substitute the preferred codon usage of channel catfish for the native sequences of the genes. To determine whether the synthetic alleles could be expressed in cells that use the standard genetic code, we introduced IAG52A[G5/CC] into a variety of heterologous cell types and tested for expression either by immunofluorescence light microscopy or Western blotting. When cloned downstream of appropriate promoters, IAG52A[G5/CC] was expressed in Escherichia coli, mammalian COS-7 cells, and channel catfish where it elicited antigen-specific immune responses. Interestingly, the localization pattern of the corresponding gene product in COS-7 cells indicated that while the protein was correctly folded, it was not present on the cell membrane, suggesting that the signal peptides required for GPI-anchor addition differ in ciliate and mammalian systems. Construction of synthetic alleles should have practical utility in the development of vaccines against Ichthyophthirius, and at the same time, provide a general method for the expression of ciliate genes in heterologous systems.