Neutralization of Rubella Vaccine Virus and Immunodeficiency-Related Vaccine-Derived Rubella Viruses by Intravenous Immunoglobulins.

The association between granulomas and vaccine-derived rubella virus (VDRV) in people with primary immune deficiencies (PID) has raised concerns about the ability of immunoglobulin (IG) preparations to neutralize VDRVs. We investigated the capacity of IG to neutralize rubella vaccine virus and four VDRV strains. As expected, the rubella vaccine virus itself was potently neutralized by IG preparations; however, the VDRV isolates from patients after intra-host evolution, 2-6 times less so. Diagnosis of immune deficiencies before possible live-virus vaccination is thus of critical importance, while IG replacement therapy can be expected to provide protection from rubella virus infection.

The occurrence of granulomas associated with vaccine derived rubella viruses (VDRV) in people with primary immune deficiencies (PID) challenges immunoglobulin (IG) preparations regarding their rubella neutralizing ability. This study confirmed potent rubella virus neutralization capacity of IG preparations and thus suggests protection of IG-treated PID patients against rubella. The study also highlights the importance of early diagnosis and timely given IG to prevent possible systemic spread of VDRV persisting locally in granulomas.

Atorvastatin exerts dual effects of lesion regression and ovarian protection in the prevention and treatment of endometriosis.

Endometriosis is a frequent, chronic, estrogen-dependent and inflammatory gynecological disease leading to pain and infertility. Clinical and metabolic studies reveal that patients with endometriosis are susceptible to hyperlipemia and lipid dysfunction, putting them at ascending risk of cardiovascular diseases. Statins constitute a group of cholesterol-lowering drugs with pleiotropic effects. A plethora of researches have proved their ability to inhibit the growth of ectopic lesions in endometriosis. However, concerns exist about their possible adverse effects on ovarian function. This study aimed to investigate the possible effect of atorvastatin on the ovarian endocrine function and fertility capacity in the prevention and treatment of endometriosis. Here, 5 mg/kg atorvastatin was intraperitoneally injected to the endometriosis mice once a day for consecutive fourteen days during and after the development of endometriotic implants. The results indicated that atorvastatin not only led to regression of the ectopic lesions, but also caused no discernible harm to the ovary for both the preventive and the therapeutic models. In addition, it elicited a protective effect on the ovarian reserve and fertility possibly by reducing inflammation in the ovary. Hence, atorvastatin could be a promising drug for endometriosis prevention and treatment.

Rubella virus-associated necrotizing neutrophilic granuloma in a patient with common variable immunodeficiency.

Patients with inborn errors of immunity (IEI) may develop granulomas in multiple organ systems including the skin. Vaccine strain rubella virus (RuV), part of the live attenuated measles, mumps, and rubella (MMR) vaccine, has been identified within these granulomas. RuV is typically found in macrophages; however, recently neutrophils have been identified as a novel cell type infected. Here, we present a case of RuV-associated cutaneous granuloma with RuV localized to neutrophils. A 46-year-old female with common variable immunodeficiency presented with verrucous papules and crusted plaques from the right knee to the distal shin of 20 years duration, associated with prior physical trauma. Biopsy specimen showed palisaded granulomas surrounding central necrosis with scattered aggregates of neutrophils. Vaccine-derived RuV was detected by molecular sequencing in lesional skin. Fluorescent immunohistochemistry with CD206, myeloperoxidase (MPO), and RV capsid (RVC) antibodies demonstrated that RuV localized to neutrophils but not macrophages. The clinical presentation, cutaneous findings, and likely presence of RVC-positive granulocytes in bone marrow provide potential support to the evolving hypothesis of persistent RuV within neutrophils contributing to chronic granulomatous inflammation in a milieu of immune dysregulation.

Intrafollicular fluid metabolic abnormalities in relation to ovarian hyperstimulation syndrome: Follicular fluid metabolomics via gas chromatography-mass spectrometry.

INTRODUCTION: Ovarian hyperstimulation syndrome (OHSS) is the most serious iatrogenic complication of ovulation stimulation during assisted reproductive technology. The main objective of this study was to investigate intrafollicular fluid metabolic change profiles of OHSS in non-ovarian etiologic infertility women (CON) and polycystic ovarian syndrome patients (PCOS). METHODS: 87 infertile women were divided into four subgroups: CON-Norm (CON with normal ovarian response), CON-OHSS (CON with OHSS), PCOS-Norm (PCOS with normal ovarian response), and PCOS-OHSS (PCOS with OHSS). The intrafollicular fluid metabolic profiles were analyzed with gas chromatography-mass spectrometry. The multivariable-adjusted conditional logistic regression was applied to assess the association of metabolites with OHSS risk. RESULTS: We identified 17 and 3 metabolites that related to OHSS risk in CON and PCOS, respectively. 13 OHSS risk-related metabolites in CON were unsaturated fatty acids, 8 of which were also the significantly altered metabolites between all PCOS and CON-Norm. CONCLUSION: Our study may shed light on the role of intrafollicular fluid metabolic abnormalities in the pathophysiology of OHSS. The findings suggested that there might be some metabolic heterogeneities underlying the development of OHSS in CON and PCOS women and indicated possible shared etiological factors in the development of PCOS and OHSS.

Case report: Persistent shedding of a live vaccine-derived rubella virus in a young man with severe combined immunodeficiency and cutaneous granuloma.

A young man with X-linked severe combined immunodeficiency developed a persistent vaccine-derived rubella virus (VDRV) infection, with the emergence of cutaneous granulomas more than fifteen years after receipt of two doses of measles-mumps-rubella (MMR) vaccine. Following nasopharyngeal swab (NP) collection, VDRV was detected by real-time polymerase chain reaction (RT-qPCR) and sequencing, and live, replication-competent VDRV was isolated in cell culture. To assess duration and intensity of viral shedding, sequential respiratory samples, one cerebrospinal fluid sample, and two urine samples were collected over 15 months, and VDRV RNA was detected in all samples by RT-qPCR. Live VDRV was cultured from nine of the eleven respiratory specimens and from one urine specimen. To our knowledge, this was the first reported instance of VDRV cultured from respiratory specimens or from urine. To assess potential transmission to close contacts, NP specimens and sera were collected from all household contacts, all of whom were immunocompetent and previously vaccinated with MMR. VDRV RNA was not detected in any NP swabs from the contacts, nor did serologic investigations suggest VDRV transmission to any contacts. This report highlights the need to understand the prevalence and duration of VDRV shedding in granuloma patients and to estimate the risk of VDRV transmission to immune and non-immune contacts.

Use of a rapid digital microfluidics-powered immunoassay for assessing measles and rubella infection and immunity in outbreak settings in the Democratic Republic of the Congo.

The Democratic Republic of the Congo (DRC) has a high measles incidence despite elimination efforts and has yet to introduce rubella vaccine. We evaluated the performance of a prototype rapid digital microfluidics powered (DMF) enzyme-linked immunoassay (ELISA) assessing measles and rubella infection, by testing for immunoglobulin M (IgM), and immunity from natural infection or vaccine, by testing immunoglobulin G (IgG), in outbreak settings. Field evaluations were conducted during September 2017, in Kinshasa province, DRC. Blood specimens were collected during an outbreak investigation of suspected measles cases and tested for measles and rubella IgM and IgG using the DMF-ELISA in the field. Simultaneously, a household serosurvey for measles and rubella IgG was conducted in a recently confirmed measles outbreak area. DMF-ELISA results were compared with reference ELISA results tested at DRC's National Public Health Laboratory and the US Centers for Disease Control and Prevention. Of 157 suspected measles cases, rubella IgM was detected in 54% while measles IgM was detected in 13%. Measles IgG-positive cases were higher among vaccinated persons (87%) than unvaccinated persons (72%). In the recent measles outbreak area, measles IgG seroprevalence was 93% overall, while rubella seroprevalence was lower for children (77%) than women (98%). Compared with reference ELISA, DMF-ELISA sensitivity and specificity were 82% and 78% for measles IgG; 88% and 89% for measles IgM; 85% and 85% for rubella IgG; and 81% and 83% for rubella IgM, respectively. Rubella infection was detected in more than half of persons meeting the suspected measles case definition during a presumed measles outbreak, suggesting substantial unrecognized rubella incidence, and highlighting the need for rubella vaccine introduction into the national schedule. The performance of the DMF-ELISA suggested that this technology can be used to develop rapid diagnostic tests for measles and rubella.

Partial hydatidiform mole pregnancy ended in full-term delivery of a normal infant: a case presentation.

BACKGROUND: Twin pregnancy with a partial hydatidiform mole (PHM) and a coexistent live fetus is extremely rare. The fetus usually has a normal karyotype. The surviving rate of the fetus till lung maturity is only about 25-40%. PHM pregnancy almost ends in abortion due to the presence of triploid embryo. Here, we report a case of PHM coexistent with a live fetus resulting in a live baby. CASE PRESENTATION: A PHM pregnancy was diagnosed by ultrasonography in a 28-year-old Chinese woman, with normal fetal morphology and mosaicism as indicated by amniocentesis. After being fully informed of the risks, the woman chose to proceed with the pregnancy and finally gave birth to a baby girl and the infant was delivered at term. A single placenta with vesicular changes and peripheral blood diploid chromosomes were observed. There were no serious maternal complications. In conclusion, the diagnosis, management, and monitoring of this condition, which is very rare in clinical practice, remain challenging. Under proper management, a PHM-combined pregnancy can still end in full-term delivery of a normal living fetus.

Association of Persistent Rubella Virus With Idiopathic Skin Granulomas in Clinically Immunocompetent Adults.

Importance: Vaccine-derived and wild-type rubella virus (RuV) has been identified within granulomas in patients with inborn errors of immunity, but has not been described in granulomas of healthy adults. Objective: To determine the association between RuV and atypical granulomatous inflammation in immune-competent adults. Design, Setting, and Participants: This case series, conducted in US academic dermatology clinics from January 2019 to January 2021, investigated the presence of RuV in skin specimens using RuV immunofluorescent staining of paraffin-embedded tissue sections, real-time reverse-transcription polymerase chain reaction, whole-genome sequencing with phylogenetic analyses, and cell culture by the US Centers for Disease Control and Prevention. Rubella immunoglobulin G, immunoglobulin M enzyme-linked immunoassay, and viral neutralization assays were performed for the sera of immunocompetent individuals with treatment refractory cutaneous granulomas and histopathology demonstrating atypical palisaded and necrotizing granulomas. Clinical immune evaluation was performed. Main Outcomes and Measures: Identification, genotyping, and culture of vaccine-derived and wild-type RuV within granulomatous dermatitis of otherwise clinically immune competent adults. Results: Of the 4 total immunocompetent participants, 3 (75%) were women, and the mean (range) age was 61.5 (49.0-73.0) years. The RuV capsid protein was detected by immunohistochemistry in cutaneous granulomas. The presence of RuV RNA was confirmed by real-time reverse-transcription polymerase chain reaction in fresh-frozen skin biopsies and whole-genome sequencing. Phylogenetic analysis of the RuV sequences showed vaccine-derived RuV in 3 cases and wild-type RuV in 1. Live RuV was recovered from the affected skin in 2 participants. Immunology workup results demonstrated no primary immune deficiencies. Conclusions and Relevance: The case series study results suggest that RuV (vaccine derived and wild type) can persist for years in cutaneous granulomas in clinically immunocompetent adults and is associated with atypical (palisaded and necrotizing type) chronic cutaneous granulomas. These findings represent a potential paradigm shift in the evaluation, workup, and management of atypical granulomatous dermatitis and raises questions regarding the potential transmissibility of persistent live RuV.

Granulomatous Dermatitis Associated With Rubella Virus Infection in an Adult With Immunodeficiency.

IMPORTANCE: Immunodeficiency-related, vaccine-derived rubella virus (RuV) as an antigenic trigger of cutaneous and visceral granulomas is a rare, recently described phenomenon in children and young adults treated with immunosuppressant agents. OBJECTIVE: To perform a comprehensive clinical, histologic, immunologic, molecular, and genomic evaluation to elucidate the potential cause of an adult patient's atypical cutaneous granulomas. DESIGN, SETTING, AND PARTICIPANTS: A prospective evaluation of skin biopsies, nasopharyngeal swabs, and serum samples submitted to the Centers for Disease Control and Prevention was conducted to assess for RuV using real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and viral genomic sequencing. The samples were obtained from a man in his 70s with extensive cutaneous granulomas mimicking both cutaneous sarcoidosis (clinically) and CD8+ granulomatous cutaneous T-cell lymphoma (histopathologically). The study was conducted from September 2019 to February 2021. MAIN OUTCOMES AND MEASURES: Identification and genotyping of a novel immunodeficiency-related RuV-associated granulomatous dermatitis. RESULTS: Immunohistochemistry for RuV capsid protein and RT-PCR testing for RuV RNA revealed RuV in 4 discrete skin biopsies from different body sites. In addition, RuV RNA was detected in the patient's nasopharyngeal swabs by RT-PCR. The full viral genome was sequenced from the patient's skin biopsy (RVs/Philadelphia.PA.USA/46.19/GR, GenBank Accession #MT249313). The patient was ultimately diagnosed with a novel RuV-associated granulomatous dermatitis. CONCLUSIONS AND RELEVANCE: The findings of this study suggest that clinicians and pathologists may consider RuV-associated granulomatous dermatitis during evaluation of a patient because it might have implications for the diagnosis of cutaneous sarcoidosis, with RuV serving as a potential antigenic trigger, and for the diagnosis of granulomatous cutaneous T-cell lymphoma, with histopathologic features that may prompt an evaluation for immunodeficiency and/or RuV.

PCOS follicular fluid derived exosomal miR-424-5p induces granulosa cells senescence by targeting CDCA4 expression.

Polycystic ovary syndrome (PCOS) is a heterogeneous reproductive disease, characterized by increased ovarian androgen biosynthesis, chronic anovulation and polycystic ovaries. The objective of this study was to identify the altered miRNA expression profiles in follicular fluid derived exosomes isolated from PCOS patients and to investigate the molecular functions of exosomal miR-424-5p. Herein, small RNA sequencing showed that twenty-five miRNAs were differentially expressed between control and PCOS group. The alterations in the miRNA profile were related to the endocrine resistance, cell growth and proliferation, cellular senescence and insulin signaling pathway. Among these differentially expressed miRNAs, we found that the expression of miR-424-5p was significantly decreased in PCOS exosomes and primary granulosa cells (GCs). Exosome-enriched miR-424-5p significantly promoted GCs senescence and suppressed cell proliferation. Similar to the results obtained in the cells transfected with miR-424-5p mimic, miR-424-5p mimic significantly decreased cell proliferation ability and induced senescence, but treatment with miR-424-5p inhibitor got the opposite results. In addition, cell division cycle associated 4 (CDCA4) gene displayed an inverse expression pattern to those of miR-424-5p, was identified as the direct target of miR-424-5p. Overexpression of CDCA4 reversed the effects of exosomal miR-424-5p on GCs via activation of Rb/E2F1 signaling pathway. These results demonstrate that exosomal miR-424-5p inhibits GCs proliferation and induces cellular senescence in PCOS through blocking CDCA4-mediated Rb/E2F1 signaling. Our findings provide new information on abnormal follicular development in PCOS.

Rubella Virus Infected Macrophages and Neutrophils Define Patterns of Granulomatous Inflammation in Inborn and Acquired Errors of Immunity.

Rubella virus (RuV) has recently been found in association with granulomatous inflammation of the skin and several internal organs in patients with inborn errors of immunity (IEI). The cellular tropism and molecular mechanisms of RuV persistence and pathogenesis in select immunocompromised hosts are not clear. We provide clinical, immunological, virological, and histological data on a cohort of 28 patients with a broad spectrum of IEI and RuV-associated granulomas in skin and nine extracutaneous tissues to further delineate this relationship. Combined immunodeficiency was the most frequent diagnosis (67.8%) among patients. Patients with previously undocumented conditions, i.e., humoral immunodeficiencies, a secondary immunodeficiency, and a defect of innate immunity were identified as being susceptible to RuV-associated granulomas. Hematopoietic cell transplantation was the most successful treatment in this case series resulting in granuloma resolution; steroids, and TNF-α and IL-1R inhibitors were moderately effective. In addition to M2 macrophages, neutrophils were identified by immunohistochemical analysis as a novel cell type infected with RuV. Four patterns of RuV-associated granulomatous inflammation were classified based on the structural organization of granulomas and identity and location of cell types harboring RuV antigen. Identification of conditions that increase susceptibility to RuV-associated granulomas combined with structural characterization of the granulomas may lead to a better understanding of the pathogenesis of RuV-associated granulomas and discover new targets for therapeutic interventions.

Diagnosis and management of heterotopic pregnancy following embryo transfer: clinical analysis of 55 cases from a single institution.

OBJECTIVE: The aims of this study were to summarize the clinical features of patients with heterotopic pregnancy (HP) following embryo transfer (ET) and explore the risk factors for miscarriage after surgery. METHODS: All patients with HP following ET treated by surgery between August 2014 and August 2015 in Chongqing Health Center for Women and Children were retrospectively reviewed. RESULTS: Fifty-five patients were identified, including 40 with tubal HP, 9 interstitial HP and 6 cornual HP. The most frequent manifestations before diagnosis was abdominal pain (29.1%), while 19 patients (34.5%) had no symptoms before diagnosis. The sensitivity of symptoms for HP was 65.5%. Gestational age at symptom onset of these patients with symptoms (n = 36) was 5.8 weeks (range 4.7-8.1). Forty-seven patients (85.5%) were suspected of HP when they received first transvaginal ultrasonography (TVS). The mean gestational age at diagnosis was 6.3 weeks (range 4.7-8.3, 16-41 days after ET). First TVS suggesting HP (P = 0.000) and first TVS performed before day 27 (P = 0.000) were two independent predictors for gestational age at diagnosis. Gestational age at surgery day was 6.7 weeks (range 5.3-10.7). Fifty-one patients (92.7%) resulted in a live birth. Gestational age at surgery day was the only independent risk factor for miscarriage in patients with HP treated by laparotomy (OR 0.003, 95% CI 0.001-0.604). CONCLUSIONS: Routine TVS at day 27 after ET could facilitate the diagnosis of HP, symptoms onset before or after day 27 are clues to early diagnosis. Prompt surgery after diagnosis may improve the prognosis of HP following ET.

Deferoxamine-mediated up-regulation of HIF-1α prevents dopaminergic neuronal death via the activation of MAPK family proteins in MPTP-treated mice.

Accumulating evidence suggests that an abnormal accumulation of iron in the substantia nigra (SN) is one of the defining characteristics of Parkinson's disease (PD). Accordingly, the potential neuroprotection of Fe chelators is widely acknowledged for the treatment of PD. Although desferrioxamine (DFO), an iron chelator widely used in clinical settings, has been reported to improve motor deficits and dopaminergic neuronal survival in animal models of PD, DFO has poor penetration to cross the blood-brain barrier and elicits side effects. We evaluated whether an intranasal administration of DFO improves the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced degeneration of dopaminergic neurons in the nigrostriatal axis and investigated the molecular mechanisms of intranasal DFO treatment in preventing MPTP-induced neurodegeneration. Treatment with DFO efficiently alleviated behavioral deficits, increased the survival of tyrosine hydroxylase (TH)-positive neurons, and decreased the action of astrocytes in the SN and striatum in an MPTP-induced PD mouse model. Interestingly, we found that DFO up-regulated the expression of HIF-1α protein, TH, vascular endothelial growth factor (VEGF), and growth associated protein 43 (GAP43) and down-regulated the expression of α-synuclein, divalent metal transporter with iron-responsive element (DMT1+IRE), and transferrin receptor (TFR). This was accompanied by a decrease in iron-positive cells in the SN and striatum of the DFO-treated group. We further revealed that DFO treatment significantly inhibited the MPTP-induced phosphorylation of the c-Jun N-terminal kinase (JNK) and differentially enhanced the phosphorylation of extracellular regulated protein kinases (ERK) and mitogen-activated protein kinase (MAPK)/P38 kinase. Additionally, the effects of DFO on increasing the Bcl-2/Bax ratio were further validated in vitro and in vivo. In SH-SY5Y cells, the DFO-mediated up-regulation of HIF-1α occurred via the activation of the ERK and P38MAPK signaling pathway. Collectively, the present data suggest that intranasal DFO treatment is effective in reversing MPTP-induced brain abnormalities and that HIF-1-pathway activation is a potential therapy target for the attenuation of neurodegeneration.

Retinoic acid stimulation of VEGF secretion from human endometrial stromal cells is mediated by production of reactive oxygen species.

It is widely accepted that vascular endothelial growth factor (VEGF) is involved in angiogenic functions that are necessary for successful embryonic implantation. We have shown that retinoic acid (RA), which is known to play a necessary role in early events in pregnancy, can combine with transcriptional activators of VEGF (e.g. TPA, TGF-ß, IL-1ß) to rapidly induce VEGF secretion from human endometrial stromal cells through a translational mechanism of action. We have now determined that this stimulation of VEGF by RA is mediated through an increased production of cellular reactive oxygen species (ROS). Results indicated that RA, but not TPA or TGF-ß, directly increases ROS production in endometrial stromal cells and that the co-stimulating activity of RA on VEGF secretion can be mimicked by direct addition of H2O2. Importantly, co-treatment of RA with TPA or TGF-ß further stimulated ROS production in a fashion that positively correlated with levels of VEGF secretion. The antioxidants N-acetylcysteine and glutathione monoethyl ester inhibited both RA + TPA and RA + TGF-ß-stimulated secretion of VEGF, as well as RA-induced ROS production. Treatment of cells with RA resulted in a shift in the glutathione (GSH) redox potential to a more oxidative state, suggesting that the transduction pathway leading to increased VEGF secretion is at least partially mediated through the antioxidant capacity of GSH couples. The specificity of this action on GSH-sensitive signalling pathways is suggested by the determination that RA had no effect on the redox potential of thioredoxin. Together, these findings predict a redox-mediated mechanism for retinoid regulation of localized VEGF secretion in the human endometrium that may be necessary for the successful establishment of pregnancy.

Retinoic acid is a cofactor for translational regulation of vascular endothelial growth factor in human endometrial stromal cells.

Vascular endothelial growth factor (VEGF) and endometrial angiogenesis play a critical role in successful embryonic implantation. Despite many studies of the effects of estrogen and progesterone on VEGF expression, its focal regulation at the site of implantation is unknown. Retinoic acid (RA) has been reported to regulate VEGF in a variety of cell types. Because localized RA synthesis occurs within the periimplantation endometrium, we tested the possibility that RA regulates VEGF production in endometrial stromal cells. Using primary and telomerase-immortalized human endometrial stromal cells, we determined that RA alone did not alter constitutive levels of VEGF production, but markedly amplified secretion when the cells were cotreated with activators of VEGF gene transcription (12-O-tetradecanoyl phorbol-13-acetate, TPA; TGF-beta; and IL-1beta). Whereas TPA or TGF-beta alone stimulated VEGF promoter activity and up-regulated mRNA levels, significant protein secretion was detected only after RA was added to the culture systems. Analysis of retinoids in secretory phase endometrial biopsies indicated that endogenous RA accumulated at concentrations sufficient to induce VEGF secretion. Polyribosome profile analysis showed that the addition of RA to transcriptional activators of VEGF shifted the translational suppressed VEGF mRNA transcripts into larger polyribosome complexes engaged in active translation. Although the precise mechanism(s) of the RA effect remains to be defined, it appears to be mediated by reactive oxygen species; the antioxidant N-acetylcysteine inhibited RA+TPA-stimulated secretion of VEGF by more than 80%. Together, our results demonstrate that in human endometrial stromal cells, RA can combine with transcriptional activators of VEGF to augment VEGF secretion through a translational mechanism of action mediated by reactive oxygen species. These findings suggest a link between the spatiotemporal changes of retinoid synthesis in the periimplantation stroma and the capacity to quickly up-regulate focal VEGF secretion needed to induce early angiogenic events of pregnancy.

Carcinogenic effects in a phenylketonuria mouse model.

Phenylketonuria (PKU) is a metabolic disorder caused by impaired phenylalanine hydroxylase (PAH). This condition results in hyperphenylalaninemia and elevated levels of abnormal phenylalanine metabolites, among which is phenylacetic acid/phenylacetate (PA). In recent years, PA and its analogs were found to have anticancer activity against a variety of malignancies suggesting the possibility that PKU may offer protection against cancer through chronically elevated levels of PA. We tested this hypothesis in a genetic mouse model of PKU (PAH(enu2)) which has a biochemical profile that closely resembles that of human PKU. Plasma levels of phenylalanine in homozygous (HMZ) PAH(enu2) mice were >12-fold those of heterozygous (HTZ) littermates while tyrosine levels were reduced. Phenylketones, including PA, were also markedly elevated to the range seen in the human disease. Mice were subjected to 7,12 dimethylbenz[a]anthracene (DMBA) carcinogenesis, a model which is sensitive to the anticancer effects of the PA derivative 4-chlorophenylacetate (4-CPA). Tumor induction by DMBA was not significantly different between the HTZ and HMZ mice, either in total tumor development or in the type of cancers that arose. HMZ mice were then treated with 4-CPA as positive controls for the anticancer effects of PA and to evaluate its possible effects on phenylalanine metabolism in PKU mice. 4-CPA had no effect on the plasma concentrations of phenylalanine, phenylketones, or tyrosine. Surprisingly, the HMZ mice treated with 4-CPA developed an unexplained neuromuscular syndrome which precluded its use in these animals as an anticancer agent. Together, these studies support the use of PAH(enu2) mice as a model for studying human PKU. Chronically elevated levels of PA in the PAH(enu2) mice were not protective against cancer.