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Purpose To examine the costs and cost savings associated with a large, urban teleretinal screening program for diabetic retinopathy (DR). Design Retrospective analysis. Participants Eighteen thousand twenty-five patients (36 050 eyes) screened via the Harris Health System (HHS) DR teleretinal screening program between June 2013 and April 2014. Methods Activity-based costing applied to the operational screening pathway was implemented to determine the cost of screening. Actual costs were calculated based on retrospective chart review and figures obtained from the HHS and Centers for Medicare and Medicaid Services. Theoretical costs of in-clinic examinations and delayed intervention were compared with actual costs of screening and treatment to determine costs savings. Main Outcome Measures Costs and cost savings in United States dollars were estimated. Results The per-patient cost of teleretinal screening itself was found to be $27.35, whereas the average total cost (factoring in treatment) per patient was determined to be $43.14. The physical examination-only and treatment-only models yielded cost savings estimates of $2 047 442.53 and $1 148 597.35, respectively. Conclusions The cost savings yielded by the HHS DR teleretinal screening program compared with conventional screening are substantial and corroborate the findings of similar studies that have analyzed teleretinal screening. Additionally, it can be presumed that there are additional indirect economic benefits resulting from earlier detection and treatment of disease.

Estimating parameters accurately in groundwater models for aquifers is challenging because the models are non-explicit solutions of complex partial differential equations. Modern research methods, such as Monte Carlo methods and metaheuristic algorithms, for searching an efficient design to estimate model parameters require hundreds, if not thousands of model calls, making the computational cost prohibitive. One method to circumvent the problem and gain valuable insight on the behavior of groundwater is to first apply a Galerkin method and convert the system of partial differential equations governing the flow to a discrete problem and then use a Proper Orthogonal Decomposition to project the high-dimensional model space of the original groundwater model to create a reduced groundwater model with much lower dimensions. The reduced model can be solved several orders of magnitude faster than the full model and able to provide an accurate estimate of the full model. The task is still challenging because the optimization problem is non-convex, non-differentiable and there are continuous variables and integer-valued variables to optimize. Following convention, heuristic algorithms and a combination is used search to find efficient designs for the reduced groundwater model using various optimality criteria. The main goals are to introduce new design criteria and the concept of design efficiency for experimental design research in hydrology. The two criteria have good utility but interestingly, do not seem to have been implemented in hydrology. In addition, design efficiency is introduced. Design efficiency is a method to assess how robust a design is under a change of criteria. The latter is an important issue because the design criterion may be subjectively selected and it is well known that an optimal design can perform poorly under another criterion. It is thus desirable that the implemented design has relatively high efficiencies under a few criteria. As applications, two heuristic algorithms are used to find optimal designs for a small synthetic aquifer design problem and a design problem for a large-scale groundwater model and assess their robustness properties to other optimality criteria. The results show the proof of concept is workable for finding a more informed and efficient model-based design for a water resource study.

Purpose: To report long-term outcomes of primary intraocular lens (IOL) placement in patients aged 7 to 24 months. Methods: This was a retrospective study of 27 consecutive patients (28 eyes) aged 7 to 24 months who underwent cataract surgery with primary IOL placement. Results: Average follow-up was 62.7 ± 41.7 months and the mean age of surgery was 14.4 ± 5.6 months. Mean final visual acuity was 1.02 ± 0.72 logMAR (20/209). Adverse events occurred in 7 eyes (25%) and included visual axis opacification in 6 eyes and pupillary block glaucoma in 1 eye. Seven patients (25.9%) required additional intraocular surgery. Strabismus was present in 19 patients (70.4%). Better stereopsis was correlated with better final acuity. Conclusions: Cataract surgery with IOL placement in patients aged 7 to 24 months is associated with few complications. Visual axis opacification is the most frequent adverse event. [ J Pediatr Ophthalmol Strabismus . 2017;54(3):149–155.]

To investigate the range of renal near-infrared spectroscopy (NIRS) measures in neonates undergoing therapeutic hypothermia for hypoxic ischemic encephalopathy (HIE) and to determine the association between renal NIRS measures and the development of acute kidney injury (AKI).A retrospective chart review was conducted of neonates with moderate to severe HIE who received therapeutic hypothermia at a tertiary care center from 2014 to 2016. Neonates had routine continuous NIRS monitoring of cerebral and renal saturation (Rsat) as part of their clinical care for 72 hours of cooling and until 24 hours after rewarming. The outcome of AKI was defined by an abnormal rate of decline of serum creatinine over the first 5 days of life. Mixed effects models determined the association between renal NIRS measures and AKI over time.Of 38 neonates with HIE undergoing cooling, 15 (39%) developed AKI. Rsat was lower than cerebral saturation during cooling (P .01), but Rsat increased over time after rewarming, while renal oxygen extraction levels decreased (P .0001). Neonates with AKI had higher Rsat levels (P .01) compared with those without AKI after 24 hours of life. Using receiver operating characteristic curves, Rsat75% by 24-48 hours predicted AKI with a sensitivity of 79% and specificity of 82% (area under the receiver operating characteristic curve = 0.76).Throughout cooling, neonates with AKI had higher Rsat measures than those without AKI. These differences may reflect lower oxygen extraction by the injured kidney. NIRS monitoring of Rsat may identify neonates with HIE at risk of developing AKI.

Background & Aims Radiofrequency ablation (RFA) is effective treatment for Barrett’s esophagus (BE). However, some patients have recurrence after complete eradication of intestinal metaplasia (CEIM). We investigated the incidence of and factors associated with BE recurrence, with or without neoplasia, after RFA and CEIM using data from the national Veterans Affairs (VA) healthcare system. Methods We performed a retrospective cohort study of Veterans with BE treated by RFA from 2005 through 2016 with follow-up endoscopy. Subjects were followed until BE recurrence, neoplasia, death until October 2016. CEIM, BE recurrence, and factors associated with recurrence were identified by review of medical records. We calculated incidence rates of BE recurrence, with and without neoplasia, after CEIM and identified predictors using Cox proportional hazards models. Results We identified 430 Veterans with BE who were treated with RFA; of these 337 achieved CEIM (78.4%). Most were men (98.6%), White (83.7%), and 66.0% had baseline dysplasia. Of those with CEIM, 98 patients (29.1%) had recurrence of BE during a total 906.0 patient-years of follow-up (median 1.9 years) after CEIM (incidence, 10.8%/patient-year). Dysplasia developed in 20 patients (2.2%/patient-year) and cancer in 3 patients (0.3%/patient-year). Baseline dysplasia (hazard ratio [HR], 1.71; 95% CI, 1.03–2.84) and long-segment BE (HR, 1.59; 95% CI, 1.01–2.51) increased risk of BE recurrence whereas treatment at high-volume RFA facilities reduced risk of BE recurrence (for quartile 4 vs quartile 1: HR, 0.19; 95% CI, 0.05–0.68). Conclusions In a nationwide VA system study of outcomes of RFA for BE, we associated baseline dysplasia, long-segment BE, and treatment at low-volume RFA centers with recurrence of BE after CEIM. The findings call for performing these procedures in high-volume centers.

Background PEP02 is a novel highly stable liposomal nanocarrier formulation of irinotecan. This randomized phase II study evaluated the efficacy and safety of single agent PEP02 compared with irinotecan or docetaxel in the second-line treatment of advanced oesophago-gastric (OG) cancer. Patients and methods Patients with locally advanced/metastatic disease who had failed one prior chemotherapy regimen were randomly assigned to PEP02 120 mg/m2, irinotecan 300 mg/m2 or docetaxel (Taxotere) 75 mg/m2 every 3 weeks. The primary end point was objective response rate (ORR). Simon's two-stage design was used and the ORR of interest was 20% (α = 0.05, type II error β = 0.10, null hypothesis of ORR was 5%). Results Forty-four patients per arm received treatment, and 124 were assessable for response. The ORR statistical threshold for the first stage was reached in all arms. In the intent-to-treat (ITT) population, ORRs were 13.6% (6/44), 6.8% (3/44) and 15.9% (7/44) in the PEP02, irinotecan and docetaxel arms, respectively. The median progression-free survival (PFS) and overall survival were similar between the trial arms. Commonest grade 3–4 adverse event reported was diarrhoea in the PEP02 and irinotecan groups (27.3% versus 18.2%). Conclusion The ORR associated with PEP02 was comparable with docetaxel and numerically greater than that of irinotecan. PEP02 warrants further evaluation in the advanced gastric cancer setting.

Delayed recovery from surgery causes personal suffering and substantial societal and economic costs. Whether immune mechanisms determine recovery after surgical trauma remains ill-defined. Single-cell mass cytometry was applied to serial whole-blood samples from 32 patients undergoing hip replacement to comprehensively characterize the phenotypic and functional immune response to surgical trauma. The simultaneous analysis of 14,000 phosphorylation events in precisely phenotyped immune cell subsets revealed uniform signaling responses among patients, demarcating a surgical immune signature. When regressed against clinical parameters of surgical recovery, including functional impairment and pain, strong correlations were found with STAT3 (signal transducer and activator of transcription), CREB (adenosine 3′,5′-monophosphate response element–binding protein), and NF-κB (nuclear factor κB) signaling responses in subsets of CD14 + monocytes ( R = 0.7 to 0.8, false discovery rate

The expression patterns of a variety of cytoskeletal antigens were studied in normal human tissues (placenta, umbilical cord, myometrium, colon, mammary gland, testis, skeletal muscle, myocardium) as well as in abnormal human tissues (palmar fibromatosis, fibrocystic disease of the mammary gland, mammary carcinoma). The immunohistochemical binding patterns of the monoclonal antibody GB 42 were compared to those of commercial antibodies directed against vimentin, desmin, smooth muscle myosin, pan actin, alpha-smooth muscle actin and gamma-smooth muscle actin. Methods applied comprised immunohistochemistry on cryostat sections and paraffin sections. Immunogold immunocytochemistry was performed on Lowicryl sections. The patterns of GB 42-binding were confirmed biochemically by SDS-PAGE and Western-blotting, and quantitative amino acid analysis. Our data suggest that the monoclonal antibody GB 42 recognizes an actin isoform which is identical to, or closely related to, gamma-smooth muscle actin. Unlike the commercially available antibody against gamma-smooth muscle actin, GB 42 does not cross-react with alpha-skeletal or alpha-cardiac actins. The GB 42-antigen is expressed in smooth muscle cells, myoepithelial cells and in later stages of differentiation of myofibroblasts, in all the tissues investigated. Throughout the development of smooth muscle cells and myofibroblasts, the appearance of the GB 42-antigen occurs after the expression of vimentin, desmin and alpha-smooth muscle actin, but prior to the expression of smooth muscle myosin. GB 42 is a reliable marker for higher stages of differentiation of smooth muscle cells and myofibroblasts.

Complement is a major mediator of tissue injury in several types of glomerulonephritis. However, no therapeutic agents that inhibit complement activation are available for human use. sCR1 (TP10, BRL 55736) is a recombinant, soluble human complement receptor 1 (CR1) molecule lacking transmembrane and cytoplasmic domains that inhibits C3 and C5 convertase activity by preferentially binding C4b and C3b. To test the efficacy of sCR1 on complement-mediated glomerulonephritis, rats were pretreated with sCR1 (60 mg/kg per day) before and during the induction of three models of complement-dependent glomerulonephritis (concanavalin A and antithymocyte serum models of proliferative glomerulonephritis, passive Heyman nephritis). Daily sCR1 and complement hemolytic activity levels were measured, and renal histology and urine protein excretion were examined. Mean serum sCR1 levels of 100 to 200 micrograms/mL were maintained with a reduction in complement hemolytic activity to less than 15% in most animals. In the antithymocyte serum model, sCR1-treated animals had significant reductions in mesangiolysis, glomerular platelet and macrophage infiltrates, and proteinuria at 48 h. In the concanavalin A model, sCR1 significantly reduced glomerular C3 and fibrin deposits, platelet infiltrates, and proteinuria at 48 h. In passive Heymann nephritis, proteinuria was also significantly reduced (199 +/- 8.5 versus 125 +/- 16 mg/day, P0.002) at 5 days. It was concluded that sCR1 significantly reduces both morphologic and functional consequences of several different types of complement-mediated glomerulonephritis and deserves evaluation as a potential therapeutic agent in complement-mediated immune glomerular disease in humans.

e21174 Background: Personalized medicine has become increasingly important in the management of oncology patients. Traditional approaches require a large numbers of unselected patients to enroll on...

237 Background: PEP02 is a novel nanoparticle liposome formulation of irinotecan (CPT-11) that has improved pharmacokinetics and tumor biodistribution of both CPT-11 and its active metabolite-SN38 compared to the free form drug. PEP02 has showed encouraging safety and efficacy in various tumor types, including significant antitumor activity in a human pancreatic cancer L3.6pl orthotopic nude mouse xenograft model. In previous phase I studies, PEP02 either alone or in combination with 5-FU/LV demonstrated prolonged disease control in 5 of 7 (71%) patients (pts) with gemcitabine (GEM)-refractory advanced pancreatic cancer (PC). This phase II study aims to evaluate PEP02 monotherapy as 2nd-line treatment in pts with metastatic, GEM-refractory PC. Methods: Pts were eligible if they had metastatic pancreatic adenocarcinoma, KPS ≥ 70, and progressed following one line of GEM-based therapy. Treatment consisted of PEP02 120 mg/m2 administered as a 90-minute infusion every 3 weeks. A Simon's 2-stage design was used with 16 pts in the first stage and 39 pts in total; primary objective was 3-month survival rate (OS3-month). Results: Between March 2009 and August 2010, 37 pts were enrolled at 3 centers in the U.S. and Taiwan. Characteristics for the first 31 evaluable pts: 13 M/18 F; age 39-82 yrs; 19 Asian/12 Caucasian, KPS 100/90/80/70: 5/14/4/8. Mean number of treatment cycles is 5 (range, 1-22). Disease control rate (minor response + stable disease >2 cycles) is 52%. 8 of 24 pts (33%) with elevated baseline CA19-9 have had >50% biomarker decline. To date, 23/31 pts (74%) have survived > 3 months, with 4 pts still alive after 1 year. Reasons for study discontinuation: 74% progressive disease, 9% drug-related toxicity, 17% other. Preliminary safety data is available for the first stage. Most common G3/4 adverse events included: fatigue (31%), neutropenia (25%), nausea/vomiting (19%), and diarrhea (13%). Conclusions: This study has already met its primary endpoint (predicted OS3-month >65%). PEP02 appears to have both activity and tolerable side effects for pts with metastatic, GEM-refractory PC, and represents a promising option for this pt population with few standard options. [Table: see text]

e13024 Background: PEP02 is a novel nanoparticle liposome formulation of irinotecan (CPT-11) that has improved PK and tumor biodistribution of CPT-11 and its active metabolite-SN38 with encouraging safety and tumor response in preclinical studies and a single-agent phase I study. The study is to define the DLT, MTD, and PK of PEP02 when in combination with high-dose fluorouracil/leucovorin (HDFL) in patients (pt) with advanced solid tumors. Methods: Pts who had failed to standard chemotherapy, ECOG PS 0-1 and adequate organ functions, no prior CPT-11, were eligible. PEP02 was given as 90 mins i.v. infusion on D1 in combination with 24-hr infusion of 5FU (2,000 mg/m2)/ LV (200 mg/m2) on D1 and D8, every 3 weeks. Cohorts of 3-6 pts were treated at 60, 80, 100, and 120 mg/m2. PK and PGx samples were collected. Results: A total of 16 pts were enrolled, with 3, 6, 5, and 2 at 60, 80, 100, and 120 mg/m2. DLTs were observed in 4 pts, including 2 each at 100 and 120 mg/m2 dose levels. DLTs were mainly G3 diarrhea...

2565 Background: PEP02 is a novel nanoparticle liposome formulation of irinotecan aiming to enhance tumor localization and improve pharmacokinetic properties of irinotecan and its active metabolite-SN38. The aims of the study are to define the dose-limiting toxicity (DLT), maximum tolerated dose (MTD) and pharmacokinetics (PK) of PEP02 in patients with advanced refractory solid tumors. Methods: Pts with advanced refractory solid tumors, ECOG PS 0–1, and adequate hematological, hepatic and renal functions were eligible. PEP02 was given as 90mins i.v. infusion, repeated every 3 weeks. The doses would have been escalated from 60, 120, 180 to 240 mg/m2 in a single-patient cohort accelerated titration design. PK samples were collected on days 1, 2, 3, 8 and 21. Results: A total of 11 pts (M/F 1/10; median age 47, range 41–67) were enrolled onto three dose levels, with 1, 6 and 4 pts at dose level I (60 mg/m2), II (120 mg/m2) and III (180 mg/m2), respectively. DLT was observed in 3 pts, including 1 at dose leve...

We have tested a panel of four monoclonal antibodies to the T cell receptor (TCR) gamma/delta heterodimer (delta TCS-1, TS-8, TCR delta-1, anti- C gamma m 1) in pregnant and non-pregnant uteri. The TCR gamma/delta complex was not detected on stromal lymphocytes, but was localized in the cytoplasm of the endometrial glandular epithelium from most pregnant uteri. These antibodies also reacted with endometrial glandular epithelium in a majority of non-pregnant uteri; the reactivity was more consistent in secretory phase glands than that in the proliferative phase of menstrual cycle. However, three different monoclonal antibodies to CD3 (OKT3, leu 4, UCHT-1) failed to show any reactivity, suggesting that the presence of the TCR gamma/delta complex was not associated with CD3 complex. The TCR gamma/delta-positive glandular epithelial cells did not react with two monoclonal antibodies to the TCR alpha/beta complex, and they were also CD4- and CD8-negative. Together with the loss of the class 1 MHC antigens from these endometrial glandular epithelial cells in early pregnancy, these data suggest that these TCR gamma/delta-bearing endometrial glandular cells may undergo phenotypic alterations under local regulation of gene expression.

Summary This study has shown that fresh amniotic epithelial cells which are negative by indirect immunofluorescence for HLA and Trf receptors become positive for both of these markers following prolonged culture. In addition, the amniotic antigens which are characteristic of fresh amniotic epithelial cells are lost and replaced by trophoblast antigens subsequent to maintenance of the cells in vitro. These observations suggest that, although the expression of HLA and Trf receptors are apparently negative in vivo, such gene products become activated and manifest in plasma membranes following exposure of the cells to culture conditions. In addition, the normal manifestation of the amnion antigens is lost following prolonged periods of culture. These findings indicate that the coding and expression of normally accepted transplantation antigens undergo a significant perturbation in vitro, and that markers of normal extra-embryonic epithelium in vivo are lost after the cells have been cultured in vitro.

The use of a fluorescence method employing propidium iodide to determine nuclear morphology of different cell types by epi-illumination in membrane immunofluorescence is described. Its usefulness is illustrated by differentiating nucleated and anuclear transferrin receptor-bearing cells as well as by simultaneously determining the viability of cell suspension without requiring a change from fluorescence microscopy, this causing no loss of visual accommodation.

GB24 is a mouse monoclonal antibody (IgG1) raised against human term placental microvilli. This antibody displayed pan-trophoblast reactivity pattern. In addition, GB24 recognized normal peripheral leukocytes and transformed cell lines (Daudi, HL-60, Jurkat, AV3, BeWo, HT-29) by using membrane immunofluorescence and flow cytometry. By SDS-polyacrylamide gel electrophoresis, this antibody immunoprecipitated two proteins of 62 kilodaltons (kDa) and 75 kDa from placental microvilli. Isoelectrofocusing analysis revealed that these two bands exhibited different isoelectric points: 4.7 for the 62 kDa protein, 4.6 and 4.4 for the two spots corresponding to the 75 kDa protein. Microvilli from different placentae revealed substantial differences regarding the intensity of the labeling of these two bands, suggesting that the antigens recognized by GB24 are probably allotypic.

The reactivities of Trop 3 and 4 monoclonal antibodies (MAbs) were studied on human term and 7-week extraembryonic membranes, adult tissues, and cell lines. Trop 3 MAb reacted with cells of chorionic villi, decidua, amniotic epithelium, and basal plate trophoblast. Trop 4 MAb reacted only with syncytiotrophoblast. On epithelium, Trop 3 MAb bound to stratified and glandular epithelium, but Trop 4 MAb reactivity was limited to basal keratinocytes. On peripheral blood mononuclear cells, Trop 3 MAb reacted with the majority of cells and Trop 4 MAb with the totality. Most cell lines were positive with both MAbs. However, one chemically induced MHC mutant was negative and another decreased its expression of Trop 3 antigen. Our results suggest Trop 3 MAb might recognize a monomorphic determinant of TLX antigens and Trop 4 is involved in cell proliferation or in cell-to-cell interaction.

Human amniochorion was studied by immunofluorescence with the use of antisera to human trophoblast, amniotic epithelium and transferrin as well as with monoclonal antibodies to beta 2M and HLA. The results showed that cytotrophoblast of amniochorion, like its counterpart in placenta, manifests a trophoblast-specific membrane marker and lacks beta 2M and HLA. This tissue also did not react with antisera to amniotic epithelium, and unlike its placental counterpart no evidence for transferrin receptors could be obtained. Amniotic epithelium was found to lack trophoblast antigens, beta 2M, HLA and transferrin receptors, but contained a plasma membrane marker not found on any other extra-embryonic tissues. These results show that extra-embryonic cells with characteristic membrane markers fail to manifest histocompatibility antigens, almost as though they were mutually exclusive. This is supported by the observation that transformed amnion epithelial cells lose their unique surface antigens, react with monoclonal anti-beta 2M and acquire transferrin receptors, raising the possibility that the histocompatibility neutrality of extra-embryonic cells is maintained by the normal insertion of specific fetal markers into their plasma membranes.

Blood clotting Factor V was studied on fixed and unfixed cryostat sections of term human placentae, basal plates, amniochorions, and first-trimester placentae by using one- and two-colour immunofluorescence. A monoclonal antibody (GB36) which reacts with cytotrophoblast was used to identify cytotrophoblast. Factor V was identified in villous cytotrophoblast of term chorionic villi and very weakly or not at all in first-trimester chorionic villi. Other extra-embryonic tissues were non-reactive with anti-Factor V. These data show that Factor V is a useful marker of term villous cytotrophoblast. Immunopathological consequences of this are considered.

Forty-six human fresh amniochorion membranes obtained at cesarean section were found to contain from 0 to 5% dead cells in the amniotic epithelial layer by direct counting and spectrophotometric analysis of trypan blue extracts. With the use of a double marker system it was discovered that many of the dead cells failed to bind the DNA-chelating fluorochrome propidium iodide but reacted with fluorescein isothiocyanate-labeled antibodies to human plasminogen. In addition, both fresh and cultured human amniotic epithelial cells that had plasma membranes damaged by either cryogenic shock or cytocentrifugation specifically bound plasminogen from serum, plasma, or amniotic fluid to cytoplasmic structures. Binding did not occur in other control proteins, but plasminogen was bound from very dilute solutions, suggesting specific and tight binding inside the cell. We propose that such plasminogen can be activated to plasmin within the cell by either plasminogen activators or lysosomal proteases and that this sets into motion a progression of events that are potentially damaging for the amniochorion, perhaps being of relevance in the pathophysiology of premature rupture of the membranes in human pregnancy.

A newborn male infant with epidermolysis bullosa letalis died at age 6 weeks of septicemia. He had many congenital ectodermal abnormalities and a rudimentary thymus. The spleen had no germinal centers and the lymph nodes lacked follicles. Cytotrophoblast of the amniochorion was sparse and the amniotic epithelium was distorted. Immunohistologic studies of amniotic epithelium revealed the unprecedented presence of trophoblast antigens. Before birth of the patient, the parents had a 5-year history of infertility; after death of the patient, the mother sustained a ruptured tubal pregnancy that necessitated blood transfusions and surgical removal of the tube. She subsequently became pregnant a third time and was delivered of a normal child. The wife shared four major histocompatibility (HLA) antigens with her husband. The parents' immunogenetic characteristics and the immunopathologic features and extraembryonic membranes of the child are considered in terms of maternal responses to trophoblast antigens.

Human promyelocytic leukemia (HL-60) and lymphoblastoid (Daudi) cells were studied: for transferrin receptors before and after induced differentiation with dimethyl sulfoxide (DMSO), sodium butyrate or retinoic acid. None of these reagents affected the morphology or presentation of receptors in Daudi cells, but many HL-60 morphologically matured to banded neutrophils and demonstrated a concomitant loss of transferrin binding, suggesting an important role for transferrin receptors in cellular differentiation.

Monoclonal antibody GB36, which was raised against human term syncytiotrophoblastic microvilli, was found to recognize a novel epithelial antigen. The antibody immunoprecipitated several membrane proteins from BeWo (choriocarcinoma) and HT-29 (colon adenocarcinoma) cells. Under non-reducing conditions, four peptides of 180, 155, 135 and 130 kDa were revealed by SDS-PAGE analysis. Three peptides of slightly higher molecular weights were revealed under reducing conditions; these three peptides had identical pI (6.2) as shown by two-dimensional gel electrophoresis. By immunofluorescence, GB36 reacted with villous cytotrophoblasts and cytotrophoblasts of chorion laeve, as well as with the basal surface of syncytiotrophoblast and amniotic epithelium. Extravillous trophoblasts of cytotrophoblastic shell in the basal plate and the cytotrophoblastic cell island were non-reactive. It is suggested that the antigen of GB36 may play a role in the polarity of epithelial cells and the adhesion of epithelial cells to extracellular matrix.

Purpose To define the dose-limiting toxicity (DLT), maximum tolerated dose (MTD) and pharmacokinetics (PK) of PEP02, a novel liposome-encapsulated irinotecan, in patients with advanced refractory solid tumors. Methods Patients were enrolled in cohorts of one to three to receive escalating dose of PEP02 in a phase I trial. PEP02, from 60 to 180 mg/m2, was given as a 90-min intravenous infusion, every 3 weeks. Results A total of 11 patients were enrolled into three dose levels: 60 (one patient), 120 (six patients) and 180 mg/m2 (four patients). DLT was observed in three patients, one at 120 mg/m2 (grade 3 catheter-related infection) and two at 180 mg/m2 (grade 4 neutropenia lasting for >3 days in one, grade 4 hematological toxicities and grade 4 diarrhea in the other). MTD was determined as 120 mg/m2. Comparing with those after free-form irinotecan in the literature, the dose-normalized PK of SN-38 (the active metabolite) after PEP02 was characterized by lower Cmax, prolonged terminal half-life and higher AUC but with significant inter-individual variation. One patient who died of treatment-related toxicity had significantly higher Cmax and AUC levels of SN-38 than those of the other three patients at 180 mg/m2. Post hoc pharmacogenetic study showed that the patient had a combined heterozygosity genotype of UGT1A1*6/*28. Two patients had objective tumor response. Conclusions PEP02 apparently modified the PK parameters of irinotecan and SN-38 by liposome encapsulation. The MTD of PEP02 monotherapy at 3-week interval is 120 mg/m2, which will be the recommended dose for future studies.

Normal IgG and purified IgG proteins of all 4 subclasses were digested with plasmin. As expected, IgG3 proteins were highly susceptible to degradation. Usually, activation with streptokinase resulted in faster and more accentuated degradation, but normal IgG was more intensely degraded by nonactivated plasmin. The presence of plasmin activators in IgG preparation might account for this observation.

A monoclonal antibody GB25 which was raised against placental syncytiotrophoblast reacted specifically with villous trophoblasts. It recognized syncytiotrophoblast, villous cytotrophoblasts of the first-trimester placentae, and a majority of the cytotrophoblasts in the chorion laeve, but not the residual cells of cytotrophoblastic shell in the basal plates and the cells in the cytotrophoblastic columns. This antibody will be useful for isolating and differentiating villous from extravillous cytotrophoblasts in the first-trimester placentae.

The expression of class I major histocompatibility complex (MHC) antigens on different regions of human amnion was studied by the avidin-biotin-complex immunoperoxidase (ABC) technique using monoclonal antibodies. In contrast to previous reports, the use of higher affinity monoclonal antibodies and the sensitive immunoperoxidase method has allowed the identification of class I MHC antigens on the amniotic epithelium. The level of expression is different between cells from various parts of the amnion, with the amniotic epithelium from the edge of the placenta consistently showing the strongest reactivity. Some of the class I MHC antigens expressed by the amnion are similar to those expressed by extravillous trophoblast cells in that both show much weaker or no reactivity with the monoclonal antibody 61D2.

Mouse hybridoma cell lines secreting monoclonal antibodies to human amnion were established. The reactivities of eight of these monoclonal antibodies (GB1, GB3, GB4, GB5, GB6, GB9, GB10 and GB11) on human skin and term extra-embryonic tissues, which included reflected amniochorions, basal plates, placentae, chorionic plates and umbilical cords, are reported. GB1, GB4, GB5, GB6, GB9 and GB11 showed various reactivity patterns on the epithelial cells of amnion, chorion and skin at different stages of differentiation. In addition, GB9 and GB11 showed extracellular reactivities; GB9 detected chorionic villi which were usually surrounded by fibrinoid and GB11 reached with fibrinoid structures in the placentae and chorion laeve. GB10 recognized connective tissues in fetal mesenchyme and adult dermis. This study demonstrates the expression of many shared antigens between tissues derived from the extra-embryonic ectoderm and adult skin. These monoclonal antibodies will provide useful tools for further investigations of epithelial differentiation and transformation.

In the human placenta, the maternal blood flows from the uterine arteries to fill the intervillous space where it is in direct contact with the syncytiotrophoblast of the chorionic villi. On the other hand, the fetal placental vascular structures form a complex network inside the villous stroma. The placental barrier which consists of trophoblast, chorionic stroma, and endothelium of the fetal blood vessels controls the major traffic of the interchange of materials between maternal and fetal blood (Boyd and Hamilton, 1970). The maturation and differentiation of the chorionic villi are related to the development of fetal blood vessels in the stroma (Fox, 1968; Kaufmann et al., 1985; Leiser et al., 1985). Abnormalities of the fetal blood vessels often lead to pathological changes in the chorionic villi (Fox, 1978). Since the placenta contains a very rich source of fetal vascular structures, it is not surprising that several monoclonal antibodies derived from a mouse which was immunized with cell membrane preparations from a full term placenta demonstrated specific reactivities to vascular structures. Although these antibodies are not specific only to placental vascular structures, they offer useful markers for the endothelium and differentiate several different vascular structures in the placenta. The reactivity of four monoclonal antibodies, GB37, GB40, GB41, and GB42 on the term human placenta is reported.

Antigen expression by fetal cells near the mother is of interest because of the possibility that some antigens may stimulate beneficial maternal immune responses or may prevent the development of harmful responses. The recent generation of monoclonal antibodies to amnion and trophoblast antigens together with the finding that class I histocompatibility antigen (HLA) expression by amnion cells can be manipulated provided an opportunity to determine if any relationships exist among expression of HLA, amnion antigens, and trophoblast antigens. The results of the present study demonstrate that: (1) class I HLA and amnion-specific antigens are co-expressed by IFN-γ-modulated amnion cells, so neither prevents expression of the other; (2) EGF enhances expression of two amnion-specific antigens; (3) amnion cells do not express transferrin receptors, placental alkaline phosphatase, or other antigens normally associated with trophoblasts following exposure to IFN-γ and/or EGF. The results demonstrate independent modulation of expression of class I HLA, two amnion-specific antigens, and some trophoblast-associated antigens on amnion epithelial cells.

Using solid-phase immunoabsorbent prepared antibody and biochemically characterized antigen, receptors for human transferrin have been identified on syncytiotrophoblastic membranes and transformed cells. Receptors with identical binding characteristics were also identified on peripheral blood lymphocytes from patients with chronic lymphatic leukemia and lymphoma, and on breast tissue biopsies from women with breast cancer. It is suggested that the binding characteristics of transferrin receptors on placental, transformed, leukemia, lymphoma and breast cancer cells are different from those found on normal cells such as reticulocytes which have a physiological requirement for iron, and that the bound apotransferrin molecules are involved in escape from immunosurveilianee.

Human promyelocytic leukemia (HL-60), Burkitt's lymphoma (Daudi) and myelogeneous leukemia (K562) cells were studied for transferrin receptors before and after being cultured with dimethyl sulfoxide, sodium butyrate or retinoic acid. Indirect immunofluorescence revealed the presence of transferrin receptors on all three cell types. However, transferrin receptors were lost from the HL-60 cells following induced differentiation to normal myelocytes, indicating a role for transferrin receptors in differentiation and transformation.

It has been shown that many types of transformed and malignant cells possess membrane receptors for transferrin (Trf), whereas most normal cells do not. This raised the possibility that these receptors might facilitate the transport of cytotoxic drugs into the receptor-bearing cells. We have linked Trf with adriamycin (Adr) and found that the conjugate inhibited the tridiated thymidine uptake of mononuclear cells in peripheral blood and bone marrow from acute myelogenous leukemia patients, while it had much less effect on receptor-negative cells. Furthermore, the inhibition was not affected when leukemic cells were incubated with the conjugate in the presence of autologous plasma, suggesting that the Trf in the plasma would not interfere with the binding of cytotoxic Trf to Trf receptors. Thus, it seems that such cytotoxic Trf conjugate may provide a valid approach for selective in vivo killing of certain neoplastic cells.

The expression of transferrin receptors, HLA-A,B,C and amnion antigens on breast biopsies from 63 patients were studied by immunofluorescence. The majority of the 36 specimens from normal breast or benign mastopathy did not express transferrin receptors, but were reactive with antibodies to HLA-A,B,C and amnion antigens. Among the specimens from patients with adenocarcinoma, 54.5% of the biopsies expressed transferrin receptors, 78.3% were non-reactive with monoclonal antibodies to common determinant of HLA-A,B,C and 63.6% failed to react with heterologous antiserum to amnion antigens. These data showed that the inverse expression of these antigens between normal and adenocarcinoma may be used as an adjunct for diagnosis of breast adenocarcinoma.

The differentiation processes of the amnion and the chorion take place simultaneously with the development of the human embryo (Boyd and Hamilton, 1970). The amnion has been considered to be derived from the trophoblasts which adjoin to the embryonic ectoderm (Bourne, 1962). At term, the amniotic epithelium of reflected and placental amnion lines the amniotic cavity, wraps around the umbilical cord and continues with the fetal skin. By using polyclonal antibodies to human amnion, three groups of antigens in common with some ectodermal tissues were identified (Hsi et al., 1984a). Recently, eight monoclonal antibodies in human amnion have been reported (Hsi and Yeh, 1986). Among them, GB3 reacted with epithelial basement membranes, but the antigen was not among the known ubiquitous components of basement membranes and GB5 recognized the junctional substances of epithelial cells. These antigens are likely to contribute to the cohesion with the subjacent tissues and to the intercellular binding of the epithelium. We report here the expression of these epithelial antigens by trophoblasts in chorion laeve and basal plate, as well as that of breast tumor cells.

The spiral arteries of the placental bed lose clearly recognizable arterial features early in pregnancy because of their invasion by extravillous trophoblast. Extensive structural alterations take place in the walls of invaded arteries, their walls containing an amorphous acidophilic “fibrinoid” matrix in which endovascular trophoblast is embedded. Such spiral arteries examined by immunofluorescence techniques in placental bed biopsy specimens obtained at cesarean section show a characteristic pattern of reaction with antisera to epithelial basement membrane amniotic antigens. These antisera have been previously demonstrated to react with the basement membrane of human amnion and of ectodemally derived epithelium such as breast ducts and skin. Interstitial trophoblast usually does not contain these antigens, and it is postulated that, following vascular invasion, endovascular trophoblast produces amniotic antigens to establish its presence. This may be of importance in maintaining the structural integrity of spiral arteries and thereby adequate maternal blood flow to the fetal-placental unit.

Significantly more peripheral blood mononuclear cells (PBM) from 39 patients with lymphoma (p less than 0.001), myeloma (p less than 0.001), or leukemia (p less than 0.001-0.025) bind transferrin (Trf) than those of 23 normals. That Trf was bound to a receptor on these cells was shown by the inhibition of these cells bound to 125I-Trf with unlabeled Trf. The paucity of Trf receptors on PBM from normals and their presence on diseased cells prompt a suggestion that Trf receptors may offer a useful approach to the diagnosis and possible treatment of some blood dyscrasias.

A protein which is recognized by an antibody to human amniotic epithelial basement membrane was identified at the basal lamina of human epidermis by immunohistology. This protein was localized at the lamina lucida of human epidermal basement membrane by immunoelectron microscopy. Studies of normal human keratinocyte cultures and epidermal wound healing suggested that the protein was probably produced by keratinocytes. By immunoblotting, a basic apparent isoelectric pH (pHiapp = 7.3) protein band of 37 kD was seen. These data indicate that this 37 kD protein, clearly different from other known basement membrane components, is present in simple and stratified epithelia of ectodermal origin, and is associated with hemidesmosomes.

A monoclonal antibody, GB17, was obtained from a hybridoma produced following murine immunization with isolated human term syncytiotrophoblastic microvilli. Immunohistological studies demonstrated the GB17 antigen to be present on syncytiotrophoblast of human term, first- and second trimester placenta, but not on human extravillous trophoblast or baboon placental trophoblast. Other normal adult human tissues and transformed cell lines tested were non-reactive with this antibody. Radio-iodinated term syncytiotrophoblastic microvillous protein was immunoprecipitated by GB17, and shown by sodium dodecyl sulfate—polyacrylamide gel electrophoresis to migrate as a single protein band at 175 kDa. The restricted tissue specificity suggests that the GB17 antigen could be useful for the development of a contragestational vaccine.

GB3 and GB5 are mouse monoclonal antibodies raised against human amnion. GB3 detects the epidermal basement membrane, and GB5 reacts with the junctional substances between the epithelial cells. These two antibodies were studied on breast tissues by indirect immunofluorescence. All (n = 8) of the normal mammary glandular basement membranes and epithelia reacted strongly with GB3 and GB5. In the intraductal carcinomas (n = 10), although nine out of ten tissues were reactive, their reactivities were greatly reduced. More strikingly, in the infiltrating carcinomas (n = 15), none of the tumor specimens were recognized by GB3 and only one out of fifteen biopsies was moderately reactive with GB5, indicating that the syntheses of the antigens of GB3 and GB5 were discontinued in the metastatic breast adenocarcinomas. These data suggest that these antigens may contribute to the interaction of the epithelium with the extracellular matrix and the intercellular binding between the epithelial cells; the loss of these antigens may facilitate the wide dissemination of tumor cells.

The presence of the first complement component around some fetal stem vessels in the chorionic villous stroma of term human placentae was first reported by Faulk and Johnson (1977) using the antiserum to Clq. This finding was later extended to the immature placenta (Johnson and Faulk, 1978). Sinha et al. (1984) showed that more anti-Clq positive fetal blood vessels and fibrinoid structures could be seen in the placentae from patients with pre-eclampsia. Although, the pathogenesis of this type of fetal stem vessels was unclear, it has been suggested that they may be mediated by immunological mechanisms (Sinha et al., 1984). Morphologically, two types of lesions of fetal stem vessels have been described, namely, fibromuscular sclerosis and obliterative endarteritis (Fox, 1978). The fibromuscular sclerosis of fetal stem arteries was often associated with villous infarction and the villi which were embedded in the fibrinous plaque. The cause of this lesion was suggested to be the cessation of fetal blood flow in these blood vessels. On the other hand, the fetal arterial obliterative endarteritis was found more often in the placentae of women with pre-eclampsia, essential hypertension or diabetes melitus. It has been proposed that it was the consequence of hypoxia of the placentae (Fox, 1967a).

Summary The specificities of antisera produced in nine rabbits to seven different preparations of human amniotic epithelium and controls were studied by immunofluorescence on cryostat sections of human amniochorions, placentae and a large collection of other tissues. Three different groups of reaction patterns were identified with the use of these antibodies, indicating the presence of at least three antigenic groupings which have been tentatively designated as amnion antigens 1 (AA1), amnion antigens 2 (AA2) and amnion antigens 3 (AA3). The AA1 group was found on the ectodermal tissues of breast ductal and corneal epithelium as well as on Hassall's corpuscles, while the AA2 group was also found on breast ductal and corneal epithelium and basal keratinocytes, but not on Hassall's corpuscles. The AA3 group was found on basement membranes of ectodermally derived epithelium, but was negative on kidney. The identification of the distribution of antigens in common with ectodermal tissues and extra-embryonic membranes suggests that human amnion and chorion may have a close embryological relationship with embryonic ectoderm and its subsequent products of organogenesis.