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Interleukin-1 receptor associated kinase 4 (IRAK4) is an essential mediator of the IL-1R and TLR signaling pathways, both of which have been implicated in multiple autoimmune conditions. Hence, blocking the activity of IRAK4 represents an attractive approach for the treatment of autoimmune diseases. The activity of this serine/threonine kinase is dependent on its kinase and scaffolding activities; thus, degradation represents a potentially superior approach to inhibition. Herein, we detail the exploration of structure-activity relationships that ultimately led to the identification of KT-474, a potent, selective, and orally bioavailable heterobifunctional IRAK4 degrader. This represents the first heterobifunctional degrader evaluated in a nononcology indication and dosed to healthy human volunteers. This molecule successfully completed phase I studies in healthy adult volunteers and patients with atopic dermatitis or hidradenitis suppurativa. Phase II clinical trials in both of these indications have been initiated.

Developing therapies for the activated B-cell like (ABC) subtype of diffuse large B-cell lymphomas (DLBCL) remains an area of unmet medical need. A subset of ABC DLBCL tumors is driven by activating mutations in myeloid differentiation primary response protein 88 (MYD88), which lead to constitutive activation of interleukin-1 receptor associated kinase 4 (IRAK4) and cellular proliferation. IRAK4 signaling is driven by its catalytic and scaffolding functions, necessitating complete removal of this protein and its escape mechanisms for complete therapeutic suppression. Herein, we describe the identification and characterization of a dual-functioning molecule, KT-413 and show it efficiently degrades IRAK4 and the transcription factors Ikaros and Aiolos. KT-413 achieves concurrent degradation of these proteins by functioning as both a heterobifunctional degrader and a molecular glue. Based on the demonstrated activity and safety of KT-413 in preclinical studies, a phase 1 clinical trial in B-cell lymphomas, including MYD88 mutant ABC DLBCL, is currently underway.

Evaluations of dogs as lung cancer detectors using breath samples have produced a variety of results, some quite promising. Breath samples are typically collected onto a substrate and stored in a sealed container when not in use, but volatile compounds dissipate when the substrate is exposed during training and evaluation sessions. Collection of appropriate samples for training and testing dogs requires significant resources and strict control of recruitment and sample collection processes. Therefore, some researchers re-use samples while training dogs. No systematic evaluation of the effect of sample re-use on dogs' training performance has been conducted, so the influence of this potentially important training factor is not known. We trained seven dogs to indicate the presence of lung cancer positive breath samples using an automated apparatus. The samples were stored at -60 °C or -80 °C. Samples from 460 individuals who were classified as positive or negative for lung cancer were used for training samples. Individual samples were presented to dogs up to four times over a period of 2 years. As sample re-use increased, sensitivity declined (-6.65, p = < .001, 95% CI [-10.56, -2.76]), specificity increased (2.87, p = .036, 95% CI [.19, 5.55]), and the dogs' bias shifted in the direction of a negative indication bias (-.094, p = < .001, 95% CI [-.149, -.39]). However, there were no significant changes in the measure associated with the detectability of the target (-0.30, p = .285, 95% CI [-.087, .26]). All observed changes in performance across sample re-use were small. Therefore, these findings suggest that sample re-use may be appropriate for training, but additional research is required to determine which factors underly changes in performance as breath samples are re-used., (Creative Commons Attribution license.)

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The role of the ribosome in the regulation of gene expression has come into increased focus. It is proposed that ribosomes are catalytic engines capable of changing their protein composition in response to environmental stimuli. Time-resolved cryo-electron microscopy (cryo-EM) techniques are employed to identify quantitative changes in the protein composition and structure of the Saccharomyces cerevisiae 80S ribosomes after shifting the carbon source from glucose to glycerol. Using cryo-EM combined with the computational classification approach, it is found that a fraction of the yeast cells' 80S ribosomes lack ribosomal proteins at the entrance and exit sites for tRNAs, including uL16(RPL10), eS1(RPS1), uS11(RPS14A/B), and eS26(RPS26A/B). This fraction increased after a change from glucose to glycerol medium. The quantitative structural analysis supports the hypothesis that ribosomes are dynamic complexes that alter their composition in response to changes in growth or environmental conditions., (© 2020 Wiley-VCH GmbH.)

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity and cell permeability to give BJP-06-005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.

The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here, we present the discovery and characterization of a pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kα/β/γ. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K's reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders., Competing Interests: Declaration of Interests J.A.M. is a member of the scientific advisory board (SAB) of 908 Devices. L.C.C. is a founder and member of the Board of Directors of Agios Pharmaceuticals and is a founder and receives research support from Petra Pharmaceuticals. These companies are developing novel therapies for cancer. N.S.G. is a founder, SAB member, and equity holder in Gatekeeper, Syros, Petra, C4, B2S, and Soltego. The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield, and Sanofi. N.S.G., T.Z., and N.P.K. are inventors on a patent application covering chemical matter in this publication owned by the Dana-Farber Cancer Institute., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

To identify protein-protein interactions and phosphorylated amino acid sites in eukaryotic mRNA translation, replicate TAP-MudPIT and control experiments are performed targeting Saccharomyces cerevisiae genes previously implicated in eukaryotic mRNA translation by their genetic and/or functional roles in translation initiation, elongation, termination, or interactions with ribosomal complexes. Replicate tandem affinity purifications of each targeted yeast TAP-tagged mRNA translation protein coupled with multidimensional liquid chromatography and tandem mass spectrometry analysis are used to identify and quantify copurifying proteins. To improve sensitivity and minimize spurious, nonspecific interactions, a novel cross-validation approach is employed to identify the most statistically significant protein-protein interactions. Using experimental and computational strategies discussed herein, the previously described protein composition of the canonical eukaryotic mRNA translation initiation, elongation, and termination complexes is calculated. In addition, statistically significant unpublished protein interactions and phosphorylation sites for S. cerevisiae's mRNA translation proteins and complexes are identified., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Covalent kinase inhibitors, which typically target cysteine residues, represent an important class of clinically relevant compounds. Approximately 215 kinases are known to have potentially targetable cysteines distributed across 18 spatially distinct locations proximal to the ATP-binding pocket. However, only 40 kinases have been covalently targeted, with certain cysteine sites being the primary focus. To address this disparity, we have developed a strategy that combines the use of a multi-targeted acrylamide-modified inhibitor, SM1-71, with a suite of complementary chemoproteomic and cellular approaches to identify additional targetable cysteines. Using this single multi-targeted compound, we successfully identified 23 kinases that are amenable to covalent inhibition including MKNK2, MAP2K1/2/3/4/6/7, GAK, AAK1, BMP2K, MAP3K7, MAPKAPK5, GSK3A/B, MAPK1/3, SRC, YES1, FGFR1, ZAK (MLTK), MAP3K1, LIMK1, and RSK2. The identification of nine of these kinases previously not targeted by a covalent inhibitor increases the number of targetable kinases and highlights opportunities for covalent kinase inhibitor development., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cyclin-dependent kinase 14 (CDK14) and other TAIRE family kinases (CDKs 15-18) are proteins that lack functional annotation but are frequent off-targets of clinical kinase inhibitors. In this study we develop and characterize FMF-04-159-2, a tool compound that specifically targets CDK14 covalently and possesses a TAIRE kinase-biased selectivity profile. This tool compound and its reversible analog were used to characterize the cellular consequences of covalent CDK14 inhibition, including an unbiased investigation using phospho-proteomics. To reduce confounding off-target activity, washout conditions were used to deconvolute CDK14-specific effects. This investigation suggested that CDK14 plays a supporting role in cell-cycle regulation, particularly mitotic progression, and identified putative CDK14 substrates. Together, these results represent an important step forward in understanding the cellular consequences of inhibiting CDK14 kinase activity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Despite recent clinical successes for irreversible drugs, potential toxicities mediated by unpredictable modification of off-target cysteines represents a major hurdle for expansion of covalent drug programs. Understanding the proteome-wide binding profile of covalent inhibitors can significantly accelerate their development; however, current mass spectrometry strategies typically do not provide a direct, amino acid level readout of covalent activity for complex, selective inhibitors. Here we report the development of CITe-Id, a novel chemoproteomic approach that employs covalent pharmacologic inhibitors as enrichment reagents in combination with an optimized proteomic platform to directly quantify dose-dependent binding at cysteine-thiols across the proteome. CITe-Id analysis of our irreversible CDK inhibitor THZ1 identified dose-dependent covalent modification of several unexpected kinases, including a previously unannotated cysteine (C840) on the understudied kinase PKN3. These data streamlined our development of JZ128 as a new selective covalent inhibitor of PKN3. Using JZ128 as a probe compound, we identified novel potential PKN3 substrates, thus offering an initial molecular view of PKN3 cellular activity. CITe-Id provides a powerful complement to current chemoproteomic platforms to characterize the selectivity of covalent inhibitors, identify new, pharmacologically addressable cysteine-thiols, and inform structure-based drug design programs.

The eukaryotic ribosomal protein RACK1/Asc1p is localized to the mRNA exit channel of the 40S subunit but lacks a defined role in mRNA translation. Saccharomyces cerevisiae deficient in ASC1 exhibit temperature-sensitive growth. Using this null mutant, potential roles for Asc1p in translation and ribosome biogenesis are evaluated. At the restrictive temperature the asc1Δ null mutant has reduced polyribosomes. To test the role of Asc1p in ribosome stability, cryo-EM is used to examine the structure of 80S ribosomes in an asc1Δ yeast deletion mutant at both the permissive and nonpermissive temperatures. CryoEM indicates that loss of Asc1p does not severely disrupt formation of this complex structure. No defect is found in rRNA processing in the asc1Δ null mutant. A proteomic approach is applied to survey the effect of Asc1p loss on the global translation of yeast proteins. At the nonpermissive temperature, the asc1Δ mutant has reduced levels of ribosomal proteins and other factors critical for translation. Collectively, these results are consistent with recent observations suggesting that Asc1p is important for ribosome occupancy of short mRNAs. The results show the Asc1 ribosomal protein is critical in translation during heat stress., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The regulatory role of the ribosome in gene expression has come into sharper focus. It has been proposed that ribosomes are dynamic complexes capable of changing their protein composition in response to environmental stimuli. MS is applied to identify quantitative changes in the protein composition of S. cerevisiae 80S ribosomes in response to different environmental stimuli. Using quantitative MS, it is found that the paralog yeast ribosomal proteins RPL8A (eL8A) and RPL8B (eL8B) change their relative proportions in the 80S ribosome when yeast is switched from growth in glucose to glycerol. By using yeast genetics and polysome profiling, it is shown that yeast ribosomes containing either RPL8A or RPL8B are not functionally interchangeable. The quantitative proteomic data support the hypothesis that ribosomes are dynamic complexes that alter their composition and functional activity in response to changes in growth or environmental conditions., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Male stress urinary incontinence (SUI) after radical prostatectomy (RP) is common. The surgical standard of care traditionally has been placement of an artificial urinary sphincter (AUS) but since its introduction the transobturator male sling has been shown to have particular unique advantages. Our aim was to assess outcomes of a consecutive series of suburethral sling insertions in men presenting with all degrees of post RP SUI., Materials and Methods: A consecutive cohort of men undergoing AdVance sling insertion following RP were studied. Parameters assessed included pre and postoperative urinary function, 24 hour pad use, quality of life (QoL) outcomes, complications and further treatments. Degree of incontinence was categorized as mild (1-2), moderate (3-5) or severe (≥ 6) depending on daily pad use. Patients were reviewed at 1, 4 and 6 months. The International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF) was used to assess symptom severity and QoL outcomes., Results: Seventy-seven patients were included, mean age 68 and mean time to sling post RP 34 (8-113) months. Preoperative degree of incontinence: mild 22%, moderate 58%, severe 20%. Fourteen percent had undergone post RP radiation therapy (RT). In total 73% experienced complete resolution of symptoms post sling, 12% significant improvement, 15% no reduction in pad use. Sixty percent with severe incontinence were classified as cured (no pad or 1 dry pad for security reasons). When patients with preoperative RT were excluded, cure rate rose to 82%. On follow up survey at 30 months (mean), the ICIQ-SF score decreased from baseline 17.7 (9-21.0) to 8.0 (0-20) (p < 0.0001), CI 95% (8-12)., Conclusions: Suburethral slings are effective and safe for all degrees of post RP incontinence, are associated with improved QoL parameters and with appropriate selection and counseling are a viable option for more severe degrees of post RP SUI.

The recent approval of covalent inhibitors for multiple clinical indications has reignited enthusiasm for this class of drugs. As interest in covalent drugs has increased, so too has the need for analytical platforms that can leverage their mechanism-of-action to characterize modified protein targets. Here we describe novel gas phase dissociation pathways which yield predictable fragment ions during MS/MS of inhibitor-modified peptides. We find that these dissociation pathways are common to numerous cysteine-directed probes as well as the covalent drugs, Ibrutinib and Neratinib. We leverage the predictable nature of these fragment ions to improve the confidence of peptide sequence assignment in proteomic analyses and explore their potential use in selective mass spectrometry-based assays.

Using affinity purifications coupled with mass spectrometry and yeast two-hybrid assays, we show the Saccharomyces cerevisiae translation initiation factor complex eukaryotic translation initiation factor 2B (eIF2B) and the very-long-chain fatty acid (VLCFA) synthesis keto-reductase enzyme YBR159W physically interact. The data show that the interaction is specifically between YBR159W and eIF2B and not between other members of the translation initiation or VLCFA pathways. A ybr159wΔ null strain has a slow-growth phenotype and a reduced translation rate but a normal GCN4 response to amino acid starvation. Although YBR159W localizes to the endoplasmic reticulum membrane, subcellular fractionation experiments show that a fraction of eIF2B cofractionates with lipid membranes in a YBR159W-independent manner. We show that a ybr159wΔ yeast strain and other strains with null mutations in the VLCFA pathway cause eIF2B to appear as numerous foci throughout the cytoplasm.

The integrity of male germ cell genome is critical for the correct progression of spermatogenesis, successful fertilization, and proper development of the offspring. Several DNA repair pathways exist in male germ cells. However, unlike somatic cells, key components of such pathways remain largely unidentified. Gametogenetin (GGN) is a testis-enriched protein that has been shown to bind to the DNA repair protein FANCL via yeast-two-hybrid assays. This finding and its testis-enriched expression pattern raise the possibility that GGN plays a role in DNA repair during spermatogenesis. Herein we demonstrated that the largest isoform GGN1 interacted with components of DNA repair machinery in the mouse testis. In addition to FANCL, GGN1 interacted with the critical component of the Fanconi Anemia (FA) pathway FANCD2 and a downstream component of the BRCA pathway, BRCC36. To define the physiological function of GGN, we generated a Ggn null mouse line. A complete loss of GGN resulted in embryonic lethality at the very earliest period of pre-implantation development, with no viable blastocysts observed. This finding was consistent with the observation that Ggn mRNA was also expressed in lower levels in the oocyte and pre-implantation embryos. Moreover, pachytene spermatocytes of the Ggn heterozygous knockout mice showed an increased incidence of unrepaired DNA double strand breaks (DSBs). Together, our results suggest that GGN plays a role in male meiotic DSB repair and is absolutely required for the survival of pre-implantation embryos.

The Tmem26 gene encodes a novel protein that we have previously shown to be regulated by hedgehog signalling in the mouse limb. We now report that Tmem26 expression is spatially and temporally restricted in other regions of the mouse embryo, most notably the facial primordia. In particular, Tmem26 expression in the mesenchyme of the maxillary and nasal prominences is coincident with fusion of the primary palate. In the secondary palate, Tmem26 is expressed in the palatal shelves during their growth and fusion but is downregulated once fusion is complete. Expression was also detected at the midline of the expanding mandible and at the tips of the eyelids as they migrate across the cornea. Given the spatio-temporally restricted expression of Tmem26, we sought to uncover a functional role in embryonic development through targeted gene inactivation in the mouse. However, ubiquitous inactivation of Tmem26 led to no overt phenotype in the resulting embryos or adult mice, suggesting that TMEM26 function is dispensable for embryonic survival.

The prevalence of tumours of the germ line is increasing in the male population. This complex disease has a complex aetiology. We examine the contribution of genetic mutations to the development of germ line tumours in this review. In particular, we concentrate on fly and mouse experimental systems in order to demonstrate that mutations in some conserved genes cause pathologies typical of certain human germ cell tumours, whereas other mutations elicit phenotypes that are unique to the experimental model. Despite these experimental systems being imperfect, we show that they are useful models of human testicular germ cell tumourigenesis.

During mouse embryogenesis, macrophage-like cells arise first in the yolk sac and are produced subsequently in the liver. The onset of liver hematopoiesis is associated with the transition from primitive to definitive erythrocyte production. This report addresses the hypothesis that a similar transition in phenotype occurs in myelopoiesis. We have used whole mount in situ hybridization to detect macrophage-specific genes expressed during mouse development. The mouse c-fms mRNA, encoding the receptor for macrophage colony-stimulating factor (CSF-1), was expressed on phagocytic cells in the yolk sac and throughout the embryo before the onset of liver hematopoiesis. Similar cells were detected using the mannose receptor, the complement receptor (CR3), or the Microphthalmia transcription factor (MITF) as mRNA markers. By contrast, other markers including the F4/80 antigen, the macrophage scavenger receptor, the S-100 proteins, S100A8 and S100A9, and the secretory product lysozyme appeared later in development and appeared restricted to only a subset of c-fms-positive cells. Two-color immunolabeling on disaggregated cells confirmed that CR3 and c-fms proteins are expressed on the same cells. Among the genes appearing later in development was the macrophage-restricted transcription factor, PU.1, which has been shown to be required for normal adult myelopoiesis. Mice with null mutations in PU.1 had normal numbers of c-fms-positive phagocytes at 11.5dpc. PU.1(-/-) embryonic stem cells were able to give rise to macrophage-like cells after cultivation in vitro. The results support previous evidence that yolk sac-derived fetal phagocytes are functionally distinct from those arising in the liver and develop via a different pathway.

Experimental evidence is presented indicating that the expression of a lacZ reporter gene driven by the HIV-1 long terminal repeat in a series of stably transfected, cloned macrophage cell lines occurs in a very small proportion of cells. The proportion of cells expressing lacZ, rather than the level of expression in each cell, is regulated by external stimuli such as LPS and phorbol ester. Based upon these and published data we propose that transcription in eukaryotic cells occurs in short pulses interspersed by long periods of inactivity of indeterminate duration. Transcriptional regulation is envisaged as involving changes in the probability rather than the rate of transcription. A probabilistic model of transcription may explain many biological phenomena, such as stem cell division and clonogenic activity, heterogeneous gene expression among clonal cell populations, retroviral latency and cell cycle progression, which appear to involve stochastic decisions.

The nonspecific immunosuppressive effect observed in pregnancy sera and mediated by two factors, immunosuppressive factors (ISF) I and II, was regulated by a third molecule termed the pregnancy-depleted immunoregulatory factor (pdIRF). Natural depletion of serum pdIRF levels during pregnancy resulted in the activation of the ISF-I and ISF-II molecules, which prior to conception existed in the serum in inactive forms. The inactive ISF-I and ISF-II molecules in male sera and in nulliparous nonpregnant female sera can be activated following the artificial selective depletion of pdIRF by absorption onto Sephacryl S-300. It is proposed that inactive complexes consisting of the ISF-I and ISF-II molecules and pdIRF and possibly involving Ca2+ and Mg2+ ions are a feature of normal sera. The pdIRF molecule has an apparent Mr of 100,000-125,000 daltons and consists of a single major polypeptide of 63,000 daltons. Equal concentrations of pdIRF are present in male and female normal sera.

The distribution of immunoglobulins in normal human endometrium throughout the menstrual cycle and in early pregnancy has been studied with an immunoperoxidase technique. In first-trimester decidua, IgG was detected within many cells of differing morphology and size. Large IgG-containing cells were often binucleate and were believed to be decidual cells. Examination of serial sections showed no kappa or lambda light-chain restriction, suggesting absorption of the immunoglobulin content. Medium-sized, irregular, IgG-containing cells were macrophages. An additional substantial population of small hyperchromatic IgG-containing cells were prominent around arterioles and adjacent to endometrial glands. From examination of adjacent sections stained with phloxine tartrazine, it was concluded that these represented endometrial granulocytes. Labelling for light chains again suggested absorption of the immunoglobulin content. In contrast, in non-pregnant endometrium immunoglobulin-containing stromal cells were uncommon, although IgG and IgA were detected in gland epithelium and secretions and in the stromal interstitium particularly in the secretory phase. These results support the notion that human endometrium lacks a classical secretory immune system and highlight the requirement for correlation between studies of cell surface markers, morphology and cell surface receptors.

During the course of a normal uncomplicated human pregnancy the mother generates an antibody response directed against determinants present on the plasma membrane of the outer fetal layer of the term placenta, the syncytiotrophoblast. The response, measured by an ELISA that utilises syncytiotrophoblast plasma membrane as the antigenic target, is predominantly IgG in nature, but with a minor contribution from IgM molecules. Maximum responses were observed during the first trimester and the levels gradually declined as the pregnancy progressed. On a population basis, this antibody response profile was mainly restricted to first and second pregnancies, although anti-trophoblast antibody responses could be detected in multiparous women but with a greatly reduced incidence compared with primipara. Mechanisms to account for these observations are discussed. Throughout, the anti-trophoblast antibody levels detected in pregnancy sera were compared with the background levels which were observed in sera obtained from males and nulliparous non-pregnant females.

The effect of gestational age and maternal parity on the development of nonspecific immunosuppressive activity in the sera of pregnant women, which inhibited the in vitro transformation of unrelated lymphocytes by phytohemagglutinin, was examined. Quantitative demonstration of this activity was dependent, in part, on the source of the lymphocytes and on the serum concentration in culture. The immunosuppressive activity became evident as the pregnancy progressed, and in late-pregnancy sera it was mediated by two factors, immunosuppressive factors (ISF) I and II with apparent Mr of 2 X 10(6) and 150,000 daltons. By analysis and comparison of different types of sera fractionated by gel filtration on Sephacryl S-300, it was evident that ISF-I and ISF-II were also present in male and nulliparous nonpregnant female sera, but in inactive forms. Hence the immunosuppressive factors did not appear to be "produced" in pregnancy, but the observed activity was a reflection of the "activation" of preexisting molecules in the serum. An accompanying report (Am J Reprod Immunol Microbiol. 1985; 9:84-90) describes the regulation of the activation event.

The anti-trophoblast antibody response generated during a normal human pregnancy and detected by a recently developed enzyme-linked immunosorbent assay, was partially characterised in terms of maternal influences, nature of the antibodies and nature of the antigenic determinants present on the syncytiotrophoblast plasma membrane. The level and incidence of the response was significantly affected by maternal parity, while the maternal ABO, but not Rhesus, blood group antigens exerted a minor influence. The antibody response was predominantly mediated by IgG molecules of the IgG1,2 and 4 subclasses. The IgG molecules existed in the maternal sera either in the form of 'free' molecules or were involved in immune complexes. The antibodies interacted with determinants that were present on all the placental membranes tested and hence are possibly organ specific. The antigenic specificities were absent from erythrocytes and peripheral blood lymphocytes.

Syncytiotrophoblast plasma membranes prepared from term placentae were selectively solubilised in non-ionic detergents. The solubilised proteins and the insoluble residue were tested in an ELISA assay for their ability to function as antigenic targets for anti-trophoblast antibodies present in normal first trimester pregnancy sera. The soluble proteins were fractionated by gel filtration and four major antigen forms were identified. The antigens were reactive with affinity purified anti-trophoblast antibody isolated from maternal sera and hence were termed maternally-recognised trophoblast antigens (MRTA); these were designated MRTA-I (Mr = 400,000 D), MRTA-II (Mr = 142,000), MRTA-III (Mr = 50,000) and MRTA-IV (Mr = 13,000). The relationship between MRTA-I, II, III and IV and antigens identified in maternal sera in the form of immune complexes is discussed.