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DNA methyltransferase and poly (ADP-ribose) polymerase inhibitors (DNMTis, PARPis) induce a stimulator of interferon genes (STING)-dependent pathogen mimicry response (PMR) in ovarian and other cancers. Here, we showed that combining DNMTis and PARPis upregulates expression of the nucleic-acid sensor NFX1-type zinc finger-containing 1 protein (ZNFX1). ZNFX1 mediated induction of PMR in mitochondria, serving as a gateway for STING-dependent interferon/inflammasome signaling. Loss of ZNFX1 in ovarian cancer cells promoted proliferation and spheroid formation in vitro and tumor growth in vivo. In patient ovarian cancer databases, expression of ZNFX1 was elevated in advanced stage disease, and ZNFX1 expression alone significantly correlated with an increase in overall survival in a phase 3 trial for therapy-resistant ovarian cancer patients receiving bevacizumab in combination with chemotherapy. RNA-sequencing revealed an association between inflammasome signaling through ZNFX1 and abnormal vasculogenesis. Together, this study identified that ZNFX1 as a tumor suppressor that controls PMR signaling through mitochondria and may serve as a biomarker to facilitate personalized therapy in ovarian cancer patients.

Lethal COVID-19 outcomes are attributed to classic cytokine storm. We revisit this using RNA sequencing of nasopharyngeal and 40 autopsy samples from patients dying of SARS-CoV-2. Subsets of the 100 top-upregulated genes in nasal swabs are upregulated in the heart, lung, kidney, and liver, but not mediastinal lymph nodes. Twenty-two of these are "noncanonical" immune genes, which we link to components of the renin-angiotensin-activation-system that manifest as increased fibrin deposition, leaky vessels, thrombotic tendency, PANoptosis, and mitochondrial dysfunction. Immunohistochemistry of mediastinal lymph nodes reveals altered architecture, excess collagen deposition, and pathogenic fibroblast infiltration. Many of the above findings are paralleled in animal models of SARS-CoV-2 infection and human peripheral blood mononuclear and whole blood samples from individuals with early and later SARS-CoV-2 variants. We then redefine cytokine storm in lethal COVID-19 as driven by upstream immune gene and mitochondrial signaling producing downstream RAAS (renin-angiotensin-aldosterone system) overactivation and organ damage, including compromised mediastinal lymph node function., Competing Interests: Competing interests statement:R.E.S. is on the scientific advisory of Miromatrix, Inc. and Lime Therapeutics and is a consultant for Alnylam, Inc. D.C.W. is on the scientific advisory boards of Pano Therapeutics, Inc., and Medical Excellent Capital.

Almost 50% of patients with myelodysplastic syndrome (MDS) are refractory to first-line hypomethylating agents (HMAs), which presents a significant clinical challenge considering the lack of options for salvage. Past work revealed that immune checkpoint molecules on peripheral myeloblasts and immune cells are up-regulated after HMA treatment. Therefore, we conducted a Phase I/II clinical trial combining guadecitabine (an HMA) and atezolizumab (an immune checkpoint inhibitor) to treat HMA-relapsed or refractory (HMA-R/R) MDS patients. This combination therapy showed median overall survival of 15.1 months relative to historical controls (4-6 months). Here, we profiled the cell composition and gene expression signatures of cells from bone marrow aspirates from trial participants with short-term (<15 months) or long-term (>15 months) survival at single-cell resolution. Long-term survivors showed a significant reduction of immunosuppressive monocytes, and an expansion of effector lymphocytes after combination therapy. Further immune profiling suggests that gamma delta T cell activation through primed dendritic cells was associated with global interferon activation in the bone marrow microenvironment of long-term survivors. Short-term survivors exhibited elevated inflammation and senescence-like gene signatures that were not resolved by combination therapy. We propose that distinct bone marrow microenvironment features, such as senescence-associated inflammation or immunosuppressive monocyte presence, could improve patient stratification for HMA and immunotherapy combinations in HMA-R/R MDS patients.

DNA methyltransferase and poly(ADP-ribose) polymerase inhibitors (DNMTis, PARPis) induce a stimulator of interferon (IFN) genes (STING)-dependent pathogen mimicry response (PMR) in ovarian (OC) and other cancers. We now show that combining DNMTis and PARPis upregulates expression of a little-studied nucleic-acid sensor, NFX1-type zinc finger-containing 1 protein (ZNFX1). We demonstrate that ZNFX1 is a novel master regulator for PMR induction in mitochondria, serving as a gateway for STING-dependent PMR. In patient OC databases, high ZNFX1 expression levels correlate with advanced stage disease. ZNFX1 expression alone significantly correlates with an increase in overall survival in a phase 3 trial for therapy-resistant OC patients receiving bevacizumab in combination with chemotherapy. In correlative RNA-seq data, inflammasome signaling through ZNFX1 correlates with abnormal vasculogenesis. ZNFX1 controls PMR signaling through the mitochondria and may serve as a biomarker to facilitate offering personalized therapy in OC patients, highlighting the strong translational significance of our findings., Competing Interests: Disclosure of Conflicts of Interest F.V.R. and S.B.B. share co-inventorship on US Provisional Patent Application Number: 61/929,680 for the concept of the combinatorial therapy. The opinions expressed in this article are the author’s own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. All other authors declare no potential conflicts of interest.

DNA methyltransferase inhibitor (DNMTi) efficacy in solid tumors is limited. Colon cancer cells exposed to DNMTi accumulate lysine-27 trimethylation on histone H3 (H3K27me3). We propose this Enhancer of Zeste Homolog 2 (EZH2)-dependent repressive modification limits DNMTi efficacy. Here, we show that low-dose DNMTi treatment sensitizes colon cancer cells to select EZH2 inhibitors (EZH2is). Integrative epigenomic analysis reveals that DNMTi-induced H3K27me3 accumulates at genomic regions poised with EZH2. Notably, combined EZH2i and DNMTi alters the epigenomic landscape to transcriptionally up-regulate the calcium-induced nuclear factor of activated T cells (NFAT):activating protein 1 (AP-1) signaling pathway. Blocking this pathway limits transcriptional activating effects of these drugs, including transposable element and innate immune response gene expression involved in viral defense. Analysis of primary human colon cancer specimens reveals positive correlations between DNMTi-, innate immune response-, and calcium signaling-associated transcription profiles. Collectively, we show that compensatory EZH2 activity limits DNMTi efficacy in colon cancer and link NFAT:AP-1 signaling to epigenetic therapy-induced viral mimicry.

Non-small-cell lung cancer remains a deadly form of human cancer even in the era of immunotherapy with existing immunotherapy strategies currently only benefiting a minority of patients. Therefore, the derivation of treatment options, which might extend the promise of immunotherapy to more patients, remains of paramount importance. Here, we define using TCGA lung squamous and lung adenocarcinoma RNAseq datasets a significant correlation between epigenetic therapy actionable interferon genes with both predicted tumor immune score generally, and CD8A specifically. IHC validation using primary sample tissue microarrays confirmed a pronounced positive association between CD8 + T cell tumor infiltration and the interferon-associated targets, CCL5 and MDA5. We next extended these findings to the assessment of clinical trial biopsies from patients with advanced non-small-cell lung cancer treated with epigenetic therapy with and without concurrent immunotherapy. These analyses revealed treatment-associated increases in both CD8 + T cell intratumoral infiltration and microenvironment CCL5 staining intensity., Competing Interests: V.A. receives research funding to her institution from Astra Zeneca and Personal Genome Diagnostics, has received research funding to Johns Hopkins University from Bristol-Myers Squibb and Delfi Diagnostics in the past 5 years and is an advisory board member for Neogenomics. V.A is an inventor on patent applications (63/276,525, 17/779,936, 16/312,152, 16/341,862, 17/047,006 and 17/598,690) submitted by Johns Hopkins University related to cancer genomic analyses, ctDNA therapeutic response monitoring and immunogenomic features of response to immunotherapy that have been licensed to one or more entities. Under the terms of these license agreements, the University and inventors are entitled to fees and royalty distributions. KAM has received consulting/advisory fees from AstraZeneca, Amgen, Janssen, Mirati Therapeutics, Daiichi Sankyo/Lilly and Puma Biotechnology, as well as Honoraria from AstraZeneca. KAM receives research funding to Johns Hopkins University from Bristol-Myers Squibb and Mirati Therapeutics. S.B.B. is on the Scientific Advisory Board for Mirati Therapeutics Inc. S.B.B. is a consultant for MDxHealth. MSP is licensed to MDxHealth in an agreement with Johns Hopkins University (JHU). S.B.B. and JHU are entitled to royalty shares received from sales. S.B.B. is an inventor of MSP which is licensed to MDxHealth in agreement with Johns Hopkins University (JHU). S.B.B. and JHU are entitled to royalty sales shares. P.A.J. is a paid consultant for Zymo Research Corporation. V.E.V. is a founder of Delfi Diagnostics, serves on the Board of Directors and as an officer for this organization, and owns Delfi Diagnostics stock, which is subject to certain restrictions under university policy. Additionally, Johns Hopkins University owns equity in Delfi Diagnostics. V.E.V. divested his equity in Personal Genome Diagnostics (PGDx) to LabCorp in February 2022. V.E.V. is an inventor on patent applications submitted by Johns Hopkins University related to cancer genomic analyses and cell-free DNA for cancer detection that have been licensed to one or more entities, including Delfi Diagnostics, LabCorp, Qiagen, Sysmex, Agios, Genzyme, Esoterix, Ventana and ManaT Bio. Under the terms of these license agreements, the University and inventors are entitled to fees and royalty distributions. V.E.V. is an advisor to Viron Therapeutics and Epitope. These arrangements have been reviewed and approved by the Johns Hopkins University in accordance with its conflict-of-interest policies., (© 2023 The Authors.)

Purpose: On the basis of preclinical evidence of epigenetic contribution to sensitivity and resistance to immune checkpoint inhibitors (ICI), we hypothesized that guadecitabine (hypomethylating agent) and atezolizumab [anti-programmed cell death ligand 1 (PD-L1)] together would potentiate a clinical response in patients with metastatic urothelial carcinoma (UC) unresponsive to initial immune checkpoint blockade therapy., Patients and Methods: We designed a single arm phase II study (NCT03179943) with a safety run-in to identify the recommended phase II dose of the combination therapy of guadecitabine and atezolizumab. Patients with recurrent/advanced UC who had previously progressed on ICI therapy with programmed cell death protein 1 or PD-L1 targeting agents were eligible. Preplanned correlative analysis was performed to characterize peripheral immune dynamics and global DNA methylation, transcriptome, and immune infiltration dynamics of patient tumors., Results: Safety run-in enrolled 6 patients and phase II enrolled 15 patients before the trial was closed for futility. No dose-limiting toxicity was observed. Four patients, with best response of stable disease (SD), exhibited extended tumor control (8-11 months) and survival (>14 months). Correlative analysis revealed lack of DNA demethylation in tumors after 2 cycles of treatment. Increased peripheral immune activation and immune infiltration in tumors after treatment correlated with progression-free survival and SD. Furthermore, high IL6 and IL8 levels in the patients' plasma was associated with short survival., Conclusions: No RECIST responses were observed after combination therapy in this trial. Although we could not detect the anticipated tumor-intrinsic effects of guadecitabine, the addition of hypomethylating agent to ICI therapy induced immune activation in a few patients, which associated with longer patient survival., (©2023 The Authors; Published by the American Association for Cancer Research.)

COVID-19, the disease caused by SARS-CoV-2, has claimed approximately 5 million lives and 257 million cases reported globally. This virus and disease have significantly affected people worldwide, whether directly and/or indirectly, with a virulent pathogen that continues to evolve as we race to learn how to prevent, control, or cure COVID-19. The focus of this review is on the SARS-CoV-2 virus' mechanism of infection and its proclivity at adapting and restructuring the intracellular environment to support viral replication. We highlight current knowledge and how scientific communities with expertize in viral, cellular, and clinical biology have contributed to increase our understanding of SARS-CoV-2, and how these findings may help explain the widely varied clinical observations of COVID-19 patients., (© 2022. The Author(s).)

DNA methyltransferase inhibitors (DNMTis) reexpress hypermethylated genes in cancers and leukemias and also activate endogenous retroviruses (ERVs), leading to interferon (IFN) signaling, in a process known as viral mimicry. In the present study we show that in the subset of acute myeloid leukemias (AMLs) with mutations in TP53 , associated with poor prognosis, DNMTis, important drugs for treatment of AML, enable expression of ERVs and IFN and inflammasome signaling in a STING-dependent manner. We previously reported that in solid tumors poly ADP ribose polymerase inhibitors (PARPis) combined with DNMTis to induce an IFN/inflammasome response that is dependent on STING1 and is mechanistically linked to generation of a homologous recombination defect (HRD). We now show that STING1 activity is actually increased in TP53 mutant compared with wild-type (WT) TP53 AML. Moreover, in TP53 mutant AML, STING1-dependent IFN/inflammatory signaling is increased by DNMTi treatment, whereas in AMLs with WT TP53 , DNMTis alone have no effect. While combining DNMTis with PARPis increases IFN/inflammatory gene expression in WT TP53 AML cells, signaling induced in TP53 mutant AML is still several-fold higher. Notably, induction of HRD in both TP53 mutant and WT AMLs follows the pattern of STING1-dependent IFN and inflammatory signaling that we have observed with drug treatments. These findings increase our understanding of the mechanisms that underlie DNMTi + PARPi treatment, and also DNMTi combinations with immune therapies, suggesting a personalized approach that statifies by TP53 status, for use of such therapies, including potential immune activation of STING1 in AML and other cancers.

Epithelial ovarian carcinomas are particularly deadly due to intratumoral heterogeneity, resistance to standard-of-care therapies, and poor response to alternative treatments such as immunotherapy. Targeting the ovarian carcinoma epigenome with DNA methyltransferase inhibitors (DNMTi) or histone deacetylase inhibitors (HDACi) increases immune signaling and recruits CD8 + T cells and natural killer cells to fight ovarian carcinoma in murine models. This increased immune activity is caused by increased transcription of repetitive elements (RE) that form double-stranded RNA (dsRNA) and trigger an IFN response. To understand which REs are affected by epigenetic therapies in ovarian carcinoma, we assessed the effect of DNMTi and HDACi on ovarian carcinoma cell lines and patient samples. Subfamily-level (TEtranscripts) and individual locus-level (Telescope) analysis of REs showed that DNMTi treatment upregulated more REs than HDACi treatment. Upregulated REs were predominantly LTR and SINE subfamilies, and SINEs exhibited the greatest loss of DNA methylation upon DNMTi treatment. Cell lines with TP53 mutations exhibited significantly fewer upregulated REs with epigenetic therapy than wild-type TP53 cell lines. This observation was validated using isogenic cell lines; the TP53 -mutant cell line had significantly higher baseline expression of REs but upregulated fewer upon epigenetic treatment. In addition, p53 activation increased expression of REs in wild-type but not mutant cell lines. These data give a comprehensive, genome-wide picture of RE chromatin and transcription-related changes in ovarian carcinoma after epigenetic treatment and implicate p53 in RE transcriptional regulation. SIGNIFICANCE: This study identifies the repetitive element targets of epigenetic therapies in ovarian carcinoma and indicates a role for p53 in this process., (©2021 American Association for Cancer Research.)

Poly(ADP ribose) polymerase inhibitors (PARPi) have efficacy in triple negative breast (TNBC) and ovarian cancers (OCs) harboring BRCA mutations, generating homologous recombination deficiencies (HRDs). DNA methyltransferase inhibitors (DNMTi) increase PARP trapping and reprogram the DNA damage response to generate HRD, sensitizing BRCA-proficient cancers to PARPi. We now define the mechanisms through which HRD is induced in BRCA-proficient TNBC and OC. DNMTi in combination with PARPi up-regulate broad innate immune and inflammasome-like signaling events, driven in part by stimulator of interferon genes (STING), to unexpectedly directly generate HRD. This inverse relationship between inflammation and DNA repair is critical, not only for the induced phenotype, but also appears as a widespread occurrence in The Cancer Genome Atlas datasets and cancer subtypes. These discerned interactions between inflammation signaling and DNA repair mechanisms now elucidate how epigenetic therapy enhances PARPi efficacy in the setting of BRCA-proficient cancer. This paradigm will be tested in a phase I/II TNBC clinical trial., Competing Interests: Competing interest statement: F.V.R. and S.B.B. are co-inventors on US Provisional Patent Application Number 61/929,680 for the concept of the combinatorial therapy.

Cancer recurrence after surgery remains an unresolved clinical problem 1-3 . Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites 4-6 . There are currently no effective interventions that prevent the formation of the premetastatic microenvironment 6,7 . Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy.

The past decade has seen the emergence of immunotherapy as a prime approach to cancer treatment, revolutionizing the management of many types of cancer. Despite the promise of immunotherapy, most patients do not have a response or become resistant to treatment. Thus, identifying combinations that potentiate current immunotherapeutic approaches will be crucial. The combination of immune-checkpoint inhibition with epigenetic therapy is one such strategy that is being tested in clinical trials, encompassing a variety of cancer types. Studies have revealed key roles of epigenetic processes in regulating immune cell function and mediating antitumour immunity. These interactions make combined epigenetic therapy and immunotherapy an attractive approach to circumvent the limitations of immunotherapy alone. In this Review, we highlight the basic dynamic mechanisms underlying the synergy between immunotherapy and epigenetic therapies and detail current efforts to translate this knowledge into clinical benefit for patients.

A minority of cancers have breast cancer gene (BRCA) mutations that confer sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis), but the role for PARPis in BRCA-proficient cancers is not well established. This suggests the need for novel combination therapies to expand the use of these drugs. Recent reports that low doses of DNA methyltransferase inhibitors (DNMTis) plus PARPis enhance PARPi efficacy in BRCA-proficient AML subtypes, breast, and ovarian cancer open up the possibility that this strategy may apply to other sporadic cancers. We identify a key mechanistic aspect of this combination therapy in nonsmall cell lung cancer (NSCLC): that the DNMTi component creates a BRCAness phenotype through downregulating expression of key homologous recombination and nonhomologous end-joining (NHEJ) genes. Importantly, from a translational perspective, the above changes in DNA repair processes allow our combinatorial PARPi and DNMTi therapy to robustly sensitize NSCLC cells to ionizing radiation in vitro and in vivo. Our combinatorial approach introduces a biomarker strategy and a potential therapy paradigm for treating BRCA-proficient cancers like NSCLC., Competing Interests: The authors declare no competing interest.

UHRF1 facilitates the establishment and maintenance of DNA methylation patterns in mammalian cells. The establishment domains are defined, including E3 ligase function, but the maintenance domains are poorly characterized. Here, we demonstrate that UHRF1 histone- and hemimethylated DNA binding functions, but not E3 ligase activity, maintain cancer-specific DNA methylation in human colorectal cancer (CRC) cells. Disrupting either chromatin reader activity reverses DNA hypermethylation, reactivates epigenetically silenced tumor suppressor genes (TSGs), and reduces CRC oncogenic properties. Moreover, an inverse correlation between high UHRF1 and low TSG expression tracks with CRC progression and reduced patient survival. Defining critical UHRF1 domain functions and its relationship with CRC prognosis suggests directions for, and value of, targeting this protein to develop therapeutic DNA demethylating agents., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Ovarian cancer is the most lethal of all gynecological cancers, and there is an urgent unmet need to develop new therapies. Epithelial ovarian cancer (EOC) is characterized by an immune suppressive microenvironment, and response of ovarian cancers to immune therapies has thus far been disappointing. We now find, in a mouse model of EOC, that clinically relevant doses of DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi, respectively) reduce the immune suppressive microenvironment through type I IFN signaling and improve response to immune checkpoint therapy. These data indicate that the type I IFN response is required for effective in vivo antitumorigenic actions of the DNMTi 5-azacytidine (AZA). Through type I IFN signaling, AZA increases the numbers of CD45 + immune cells and the percentage of active CD8 + T and natural killer (NK) cells in the tumor microenvironment, while reducing tumor burden and extending survival. AZA also increases viral defense gene expression in both tumor and immune cells, and reduces the percentage of macrophages and myeloid-derived suppressor cells in the tumor microenvironment. The addition of an HDACi to AZA enhances the modulation of the immune microenvironment, specifically increasing T and NK cell activation and reducing macrophages over AZA treatment alone, while further increasing the survival of the mice. Finally, a triple combination of DNMTi/HDACi plus the immune checkpoint inhibitor α-PD-1 provides the best antitumor effect and longest overall survival, and may be an attractive candidate for future clinical trials in ovarian cancer., Competing Interests: Conflict of interest statement: S.B.B. and C.A.Z. have a collaborative research agreement with Janssen. V.B., K.R.W., G.S.C., and K.E.B. are employed by Janssen.

Combining DNA-demethylating agents (DNA methyltransferase inhibitors [DNMTis]) with histone deacetylase inhibitors (HDACis) holds promise for enhancing cancer immune therapy. Herein, pharmacologic and isoform specificity of HDACis are investigated to guide their addition to a DNMTi, thus devising a new, low-dose, sequential regimen that imparts a robust anti-tumor effect for non-small-cell lung cancer (NSCLC). Using in-vitro-treated NSCLC cell lines, we elucidate an interferon α/β-based transcriptional program with accompanying upregulation of antigen presentation machinery, mediated in part through double-stranded RNA (dsRNA) induction. This is accompanied by suppression of MYC signaling and an increase in the T cell chemoattractant CCL5. Use of this combination treatment schema in mouse models of NSCLC reverses tumor immune evasion and modulates T cell exhaustion state towards memory and effector T cell phenotypes. Key correlative science metrics emerge for an upcoming clinical trial, testing enhancement of immune checkpoint therapy for NSCLC., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Epigenetic therapy is emerging as a potential therapy for solid tumors. To investigate its mechanism of action, we performed integrative expression and methylation analysis of 63 cancer cell lines (breast, colorectal, and ovarian) after treatment with the DNA methyltransferase inhibitor 5-azacitidine (AZA). Gene Set Enrichment Analysis demonstrated significant enrichment for immunomodulatory pathways in all three cancers (14.4-31.3%) including interferon signaling, antigen processing and presentation, and cytokines/chemokines. Strong upregulation of cancer testis antigens was also observed. An AZA IMmune gene set (AIMs) derived from the union of these immunomodulatory pathway genes classified primary tumors from all three types, into "high" and "low" AIM gene expression subsets in tumor expression data from both TCGA and GEO. Samples from selected patient biopsies showed upregulation of AIM genes after treatment with epigenetic therapy. These results point to a broad immune stimulatory role for DNA demethylating drugs in multiple cancers.

The Predictive Safety Testing Consortium's first regulatory submission to qualify kidney safety biomarkers revealed two deficiencies. To address the need for biomarkers that monitor recovery from agent-induced renal damage, we scored changes in the levels of urinary biomarkers in rats during recovery from renal injury induced by exposure to carbapenem A or gentamicin. All biomarkers responded to histologic tubular toxicities to varied degrees and with different kinetics. After a recovery period, all biomarkers returned to levels approaching those observed in uninjured animals. We next addressed the need for a serum biomarker that reflects general kidney function regardless of the exact site of renal injury. Our assay for serum cystatin C is more sensitive and specific than serum creatinine (SCr) or blood urea nitrogen (BUN) in monitoring generalized renal function after exposure of rats to eight nephrotoxicants and two hepatotoxicants. This sensitive serum biomarker will enable testing of renal function in animal studies that do not involve urine collection.

Two structurally distinct types (A, B) of microscopic sarcocysts were found in muscles of 4 of 5 feral skunks. Type A sarcocysts had sarcocyst walls of up to 6 microm thick. The villar protrusions (Vp) on the sarcocyst wall were up to 5 microm long. The Vp were constricted at the base, expanded in the middle, and had a blunt tip. Numerous microtubules were present in the Vp and in the granular layer. Bradyzoites were up to 11 microm long and up to 3.2 microm wide. Based on the distinctiveness of the Vp, a new name, Sarcocystis mephitisi is proposed for type A sarcocysts. Type B sarcocysts had a relatively thin (approximately 1-2 microm thick) sarcocyst wall and the Vp were slender and tapered toward the tip. These sarcocysts were structurally similar to S. neurona sarcocysts. A Toxoplasma gondii-like tissue cyst was found in a section of tongue of 1 of the 4 skunks.

The effect of anodization on passive dissolution of titanium was studied by measuring titanium levels in peritoneal leukocytes and tissues of laboratory animals with titanium plates implanted into the peritoneal cavity. Fifteen Sprague-Dawley rats were assigned randomly to three treatment groups of five animals. One group served as controls, the other two groups had an anodized or an unanodized implant placed in the left paracolic gutter. Peritoneal lavage samples and blood samples, organ tissues and tissue surrounding the implants, were removed for histologic examination and titanium levels. Titanium was not detected in any distant organs or in the peritoneal lavage fluid. The capsular tissues surrounding the implants contained titanium at levels ranging from 2610 to 16786 ng/g for unanodized plates, and 888-5933 ng/g for anodized plates. The titanium levels within the peritoneal leukocytes of animals with unanodized implants were significantly elevated (P = 0.01) over time, as compared with controls. The level of titanium in the peritoneal leukocytes of animals with anodized implants was not significantly elevated when compared with controls. Titanium levels in the trace range, as measured in the capsular tissues, are likely a result of corrosion. Surface treatment of titanium by anodization reduces passive dissolution.

Equine protozoal myeloencephalitis (EPM) was clinically diagnosed in a 20-year-old horse with severe ataxia. The cerebrospinal fluid was positive for Sarcocystis neurona antibodies by western blot. The horse was administered corticosteroids to facilitate in vitro culture of S. neurona from its spinal cord following necropsy. Microscopic lesions of EPM were present in the brain and in the spinal cord, including multifocal inflammatory cellular infiltrates and several large groups of protozoa. Immunohistochemical, and light and electron microscopic examinations revealed that the protozoa were Neospora caninum and not S. neurona. The protozoa divided by endodyogeny, tachyzoites had rhoptries, and organisms reacted specifically to N. caninum antibodies. Veterinarians should be aware of increasing diagnosis of N. caninum as another etiological agent responsible for the lesions of EPM.

Cryptosporidial infection was diagnosed histologically in the small intestine of a big brown bat (Eptesicus fuscus) from Oregon. This is the first report of cryptosporidiosis in a bat.

Objectives: To measure coagulation factor VIII:coagulant (F.VIII:C) and C1-esterase inhibitor (C1-INH), hemostasis-associated acute-phase reactant proteins and coagulation factors VII (F.VII), IX (F.IX), and X (F.X), hemostasis proteins not associated with an acute-phase response, in a select population of horses with colic and hemostasis abnormalities, and presumed to have acute-phase changes. To compare these values and other routine hemostasis test results in the horses with colic with values for a population of healthy horses. To correlate the values of known equine acute-phase reactants, F.VIII:C and fibrinogen, to those of other tests of hemostasis. To identify hemostasis-associated acute-phase reactant proteins and gain insights into the effects the acute-phase response has on hemostatic abnormalities in horses with colic syndrome., Sample Population: 54 plasma samples from horses with colic attributable to inflammatory (n = 39) or strangulating (n = 15) intestinal disorders., Procedure: Plasma samples were evaluated for activities of F.VII, F.VIII:C, F.IX, F.X, C1-INH, antithrombin III, protein C, plasminogen, and alpha 2-antiplasmin (alpha 2AP); fibrinogen concentration; and prothrombin (PT) and activated partial thromboplastin (APTT) times., Results: Horses with colic had significantly higher fibrinogen concentration, greater alpha 2AP and protein C activities, and longer PT and APTT than did healthy horses. Horses with colic also had significantly lower mean F.VII activity than did healthy horses. Significant positive correlations between fibrinogen concentration and F.VIII:C, C1-INH, and alpha 2AP values, and between F.VIII:C activity and fibrinogen, C1-INH, alpha 2AP, and plasminogen values were identified., Conclusions: An acute-phase response contributes to changes observed in coagulation proteins in horses with colic attributable to inflammatory and strangulating intestinal disorders. The data suggest that plasminogen, alpha 2AP, and C1-INH, should be considered equine acute-phase proteins.

Objectives: To adapt manual human chromogenic assays for coagulation factors VII (F.VII), VIII:coagulant (F.VIII:C), IX (F.IX), and X (F.X), and C1-esterase inhibitor (C1-INH) for use with an automated analyzer, and to measure the activity of these proteins in horses., Animals: 10 healthy horses were used to determine ranges for the assays. Pooled plasma for standards was collected from an additional 20 healthy horses., Procedure: A computer-assisted analyzer was programmed from the manual method for commercially available human F.VII, F.VIII:C, F.IX, F.X, and C1-INH chromogenic assay kits. Standards were prepared from pooled citrated equine plasma for the F.VII, F.VIII:C, and F.X assays, and from commercial pooled citrated human plasma for F.IX and C1-INH assays., Results: Mean +/- SD activities in citrated equine plasma from 10 horses were 226 +/- 19% for F.VII; 209 +/- 31% for F.VIII:C; 149 +/- 38% for F.IX; 88 +/- 12% for F.X; and 18.4 +/- 8.4% for C1-INH. Intra-assay coefficients of variation (CV) were 5.3% for F.VII; 2.1% for F.VIII:C; and 3.0% for C1-INH. Interassay CV were 5.7% for F.VII; 7.4% for F.VIII:C; 3.8% for F.IX; 14.4% for F.X; and 22.0% for C1-INH., Conclusions: Human chromogenic assay kits can be automated and used to measure F.VII, F.VIII:C, F.IX, F.X, and C1-INH activities in citrated equine plasma., Clinical Relevance: Human chromogenic assays can be routinely used to measure F.VII, F.VIII:C, F.IX, F.X, and C1-INH in horses, and may be useful in evaluation of horses with disorders of hemostasis.

Sarcocysts of an unidentified Sarcocystis species were found in sections of skeletal muscles of a black bear (Ursus americanus) from North Carolina. Two sarcocysts in a section measured 45 x 37.5 microm and 67.5 x 50 microm and had a thin (<2 microm) sarcocyst wall. The villar protrusions on the cyst wall were up to 2 microm long and up to 0.7 microm wide. The bradyzoites were approximately 6 X 2.5 microm in size. This is the first report of muscular Sarcocystis in a bear.

This paper describes Neospora caninum-associated hydrocephalus in an aborted Hereford bovine fetus of 7 months' gestational age. Numerous tachyzoites were observed in areas of the cerebrum with lesions of non-suppurative necrotizing encephalitis.

Acute generalized sarcocystosis was diagnosed in a capercaillie (Tetrao urogallus) from Finland. Microscopic lesions were seen in the heart, lungs, spleen, liver, and brain. Protozoa were found in all organs, especially in the lungs and spleen. Only asexual stages were observed. The parasite divided by endopolygeny. Schizonts were usually 10 microm wide and up to 55 microm long. Merozoites are 3-4 microm long and 1.5-2.0 microm wide. Sarcocysts and sexual stages are unknown. The parasite was considered to be a species of Sarcocystis with an unknown life cycle. This is the first report of acute sarcocystosis in capercaillie from Finland.

Objectives: To adapt and characterize a human ELISA kit to quantify thrombin-antithrombin III (TAT) complexes in horses, and to evaluate TAT as a marker for hypercoagulation in horses., Animals: 29 clinically normal horses used as controls, and 4 ill horses used to evaluate assay for known causes of hypercoagulation., Procedure: A commercially available human sandwich-type ELISA kit with 2 antibodies against human thrombin and antithrombin III that bind selectively to their corresponding TAT antigenic sites was used. Equine TAT standards were made from purified equine thrombin and antithrombin III. Proteins diluted in a phosphate-buffered saline solution containing 0.1% Tween and 1 U of heparin/ ml were used to establish standard curves. Reference intervals for TAT concentration in citrated equine plasma, and intra- and interassay coefficients of variation were determined., Results: Mean +/- SD values were 3.95 +/- 1.93 micrograms/L, with median of 3.18 micrograms/L and range of 1.95 to 9.03 micrograms/ L. One horse with cecal perforation had TAT concentration of 174.30 micrograms/L, and a horse infused IV with endotoxin had TAT concentration of 62.98 micrograms/L 12 hours after infusion., Conclusions: The data suggest that human TAT ELISA kits can be used to measure TAT concentration in citrated equine plasma, and that TAT is a marker for hypercoagulation in horses., Clinical Relevance: Assays for equine TAT many help to further characterize the hypercoagulable state in horses.

Objectives: To evaluate new ELISA for measurement of thrombin-antithrombin III (TAT) concentration, and to correlate the values to other tests of hemostasis in horses with colic., Design: Plasma TAT concentration and 8 other hemostasis analytes were measured in horses with colic at hospital admission and during the next 4 days. Retrospectively, data were analyzed by outcome, broad-category diagnosis, and clinical management, and for correlation between TAT and other assays., Animals: 100 horses with colic., Procedure: Plasma samples were evaluated for TAT, fibrinogen, and fibrin degradation products concentrations; antithrombin III (ATIII), protein C, alpha 2-antiplasmin, and plasminogen activities; prothrombin time (PT); and activated partial thromboplastin time., Results: Changes were indicative of a hypercoagulable state, most severe in nonsurviving horses, characterized by increased TAT concentration; decreased ATIII, protein C, and plasminogen activities; and increased PT. Nonsurvivors had significantly increased TAT concentration compared with that in survivors, without regard to sample collection time; however, compared over time, TAT was significantly increased only at admission. Highest TAT concentration was in nonsurvivors with inflammatory intestinal lesions. There was significant negative correlation between TAT and ATIII, protein C, alpha 2-antiplasmin, and plasminogen values, and significant positive correlation between TAT and PT, and fibrin degradation products values., Conclusions: Plasma TAT reflects the current state of coagulation system activation and is a good assay for early diagnosis of the hypercoagulable state in horses with the most severe forms of colic., Clinical Relevance: Measurement of equine TAT provides further information to characterize the hypercoagulable state in horses to aid in case management.

Neospora canium tachyzoites and tissue cysts were found in tissues of a goat fetus aborted after 3.5 months of gestation. The fetus had hydrocephalus and a hypoplastic cerebellum. The predominant lesion in the fetus was severe encephalitis associated with numerous N canium tissue cysts. Parasites in fetal tissues reacted positively with N caninum antibodies in immunohistochemical tests. The doe was clinically normal and had a 1:800 antibody titer to N caninum as determined by use of an indirect fluorescent antibody test 9 months after abortion. Five of 77 other does from this herd also had indirect fluorescent antbody titers to N caninum that were > or = 1:100.

Eight tests of hemostasis were measured in 233 horses with colic. Blood samples were obtained at admission and for 4 consecutive days of hospitalization. Data were analyzed retrospectively by outcome, by broad-category diagnosis group, by small intestinal disorder, and by smaller categories for comparing specific diseases. Nonsurviving horses and horses with the most severe forms of intestinal ischemia had changes interpreted as hypercoagulative, the intensity of which was increased on the first and second mornings (sample times 2 and 3) after admission, when most significant differences for results of specific tests were detected. Nonsurvivors had decreased antithrombin III activity and prolonged prothrombin and activated partial thromboplastin times; those with strangulating obstructions also had decreased protein C and plasminogen activities. During hospitalization and with survival, these changes tended to reverse. In most horses, regardless of diagnosis or outcome, concentration of fibrin degradation products and fibrinogen, and alpha 2-antiplasmin activity increased over time. Whether these changes reflected specific effects of colic or of the acute-phase response was not determined. In comparisons of small intestinal disorders (proximal enteritis, strangulations, and impactions), diagnostically distinguishing features were not found. Likewise, in comparisons of specific diseases (small vs large intestinal impaction, proximal enteritis vs colitis, small vs large intestinal obstruction), diagnostically distinguishing features were not found.

Three calves (Nos. 1, 2 = 7 days old; No. 3 = 21 days old) were inoculated subcutaneously with virulent Rift Valley fever (RVF) virus. All calves became viremic and clinically ill, but the two 7-day-old calves were moribund and were euthanatized subsequently on post-inoculation day (PID) 3. Highest viral titers were measured in the serum, with lesser concentrations in the brain, heart, spleen, and liver of these animals. Viral antigens were detected by immunohistochemical analysis only in the livers, where positive staining was localized in coalescing foci of hepatocellular necrosis. The 21-day-old calf appeared to recover after viremia and pyrexia but became lethargic and ataxic and was euthanatized on PID 9. The calf was no longer viremic, and RVF virus was isolated only from the brain. Microscopic examination of the central nervous system revealed diffuse perivascular infiltrates of lymphocytes and macrophages, multifocal meningitis, and focal areas of neuronal necrosis and aggregates of macrophages, lymphocytes, and neutrophils throughout all regions of the brain and cervical spinal cord. There was positive immunohistochemical staining for viral antigens within the cytoplasm of neurons and glial cells throughout the central nervous system. Thus, RVF virus can cause encephalomyelitis in calves, and the specific virologic diagnosis can be made by immunohistochemical localization of viral antigens in formalin-fixed tissues.

Sarcocystis neuronan n. sp. is proposed for the apicomplexan taxon associated with myeloencephalitis in horses. Only asexual stages of this parasite presently are known, and they are found within neuronal cells and leukocytes of the brain and spinal cord. The parasite is located in the host cell cytoplasm, does not have a parasitophorous vacuole, and divides by endopolygeny. Schizonts are 5-35 microns x 5-20 microns and contain 4-40 merozoites arranged in a rosette around a prominent residual body. Merozoites are approximately 4 x 1 micron, have a central nucleus, and lack rhoptries. Schizonts and merozoites react with Sarcocystis cruzi antiserum but not with Caryospora bigenetica. Toxoplasma gondii, Hammondia hammondi, or Neospora caninum antisera in an immunohistochemical test.

Two of 8 littermate Rottweiler dogs developed persistent diarrhea at 6.5 weeks of age. Dog 1 was euthanatized at 14 weeks of age and had hepatitis characterized by necrosis and mixed leukocyte infiltrations in association with a previously unrecognized Sarcocystis-like protozoon. The organism was free in the hepatocyte cytoplasm without a parasitophorous vacuole, had divided by schizogony, and stained with anti-Sarcocystis serum, but did not stain with anti-Toxoplasma gondii or anti-Neospora caninum serum in an immunohistochemical test. Dog 2 was euthanatized at 10 weeks of age. This dog had large necrotic, hemorrhagic mesenteric lymph nodes. Numerous T gondii tachyzoites were observed in association with these lesions. The organism divided by endodyogeny and stained specifically with anti-T gondii serum.