Jerry Vockley

Enhanced Defense against Pneumocystis carinii Mediated by a Novel Dectin-1 Receptor Fc Fusion Protein

Pneumocystis carinii (PC) pneumonia is a leading opportunistic infection found among HIV-infected individuals worldwide. Although CD4+ T cell deficiency clearly correlates with susceptibility to PC pneumonia, murine models of disease indicate that PC-directed Abs may prevent infection and/or inhibit growth of existing PC within the lungs. Recognition of PC by alveolar macrophages involves the β-glucan receptor Dectin-1 and macrophage effector function against PC is enhanced by Abs derived from PC-vaccinated hosts. We developed a fusion protein consisting of the extracellular domain of Dectin-1 linked to the Fc portion of murine IgG1, which we hypothesized would enhance host recognition and opsonic phagocytosis of PC. The recombinant protein, Dectin-Fc, is dimeric and the Ag recognition site identifies β-1,3 glucan linkages specifically and with high affinity (KD = 2.03 × 10−7 M). Dectin-Fc enhances RAW264.7 macrophage recognition of the β-glucan containing particulate zymosan in an FcγRII- and FcγRIII-dependent manner and preopsonization of PC organisms with Dectin-Fc increased alveolar and peritoneal macrophage-dependent killing of PC. SCID mice treated with a replication incompetent adenoviral vector expressing Dectin-Fc had attenuated growth of PC within the lungs, overall decreased PC lung burden, and diminished correlates of PC-related lung damage relative to SCID mice receiving a control vector. These findings demonstrate that targeting PC β-glucan with Dectin-Fc enhances host recognition and clearance of PC in the absence of B and T cells, and suggest that FcγR-based targeting of PC, via cell wall carbohydrate recognition, may promote resistance against PC pneumonia in the immunodeficient host.

Liver transplantation for maple syrup urine disease: A global domino effect.

In this issue of the journal, Mohan et al. describe a successful case series of two infants receiving living unrelated transplants for maple syrup urine disease (MSUD) and subsequently donating their liver, by domino technique, to two other children with chronic liver disease (1). After protocols to safely transplant children and adults with MSUD were initiated (2), more than 60 patients have been successfully transplanted at our center (3), and more worldwide. MSUD has become an elegant example of liver transplantation (LT) as gene replacement for selected metabolic diseases, and while it may be the most recent metabolic condition to be phenotypically cured by LT on a relatively large scale, it certainly will not be the last (4). Deceased donor LT and live donor liver transplant (LDLT) were the first procedures used for patients with MSUD, but more recently, MSUD LT has expanded to include living related liver (LRLT) (5–7) transplants from heterozygous donors. Furthermore, utilizing the MSUD liver as a donor organ for non-MSUD transplant recipients highlights the use of transplant therapies in the local context: combining the issues of access to medical care, donor availability, and technical expertise to provide the best care to as many patients as possible. The successful reported case experience of Mohan et al. highlights, as we have emphasized, that children face significant variability worldwide in access to prompt and efficient metabolic care or appropriate metabolic formulas as well as challenges in access to deceased donor transplantation. For these children, MSUD LT and in particular LDLT or LRLT may be a new path forward (8). Careful monitoring and long-term follow-up will help to confirm the progress that has been described in MSUD LT. Recently, Feier et al. have evaluated metabolic follow-up on four children who underwent LRLT. All four children were free of protein restriction and have not developed any episode of hyperleucinosis. Interestingly, however, we noted that mean plasma leucine:isoleucine:valine concentration ratios after living related transplant for MSUD were different than described in deceased donor transplantation (9), suggesting a possible need for valine supplementation post-transplant and need for long-term monitoring of amino acids and molar ratios post-transplant, particularly in times of intercurrent illness. The unique pathophysiology of branched chain amino acid metabolism has also led to the utility of MSUD explanted livers as domino livers to benefit non-MSUD patients in need of liver transplant (10). Unlike domino transplant using other explanted livers such as from amyloidosis patients (11), domino MSUD LT has not resulted in transmission of MSUD and is not expected to do so, even over the long term. The technical expertise and team coordination required to carry out domino transplantation should not be underestimated. Although beyond the scope of this editorial, the standardization of domino transplant technique presents an important opportunity in the transplant field. In our own protocol, heparinization of the donor MSUD recipient, careful decision making in regard to hepatectomy technique, and use of hepatic venoplasty or venous reconstructions only when necessary have led to excellent results. Others have reported transplant in even younger MSUD patients (6) or domino MSUD from even younger recipients (Mureo Kasahara, personal communication) than the cases reported by Mohan et al. Indeed, transplantation in very young MSUD children may give the best opportunity for preservation of neurodevelopmental outcomes (12). Also, the technical challenges in domino transplantation of extremely young MSUD grafts may be mitigated by microvascular techniques, as has been reported (13).

Medium chain acyl-CoA dehydrogenase deficiency in a premature infant

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is identified by newborn screening (NBS). The natural history of MCADD includes metabolic decompensation with hypoglycemia, hyperammonemia, seizures, coma, and death. NBS enables expectant management thus severe symptoms are rare in managed patients. We report premature birth of an MCADD affected infant and resultant management challenges. Nutritional support advanced from parenteral nutrition at 24 hours to enteral feeds. A NBS sample was collected day 2, positive results for MCADD was reported day six, and diagnostic tests were performed day seven. Lab results confirmed MCADD; however, representation of pathologic analytes was so extreme that ingestion of medium chain triglycerides was suspected and subsequently confirmed. Diet was adjusted and reflected in moderation of pathologic analytes. This case emphasizes the need for prompt review NBS results in premature infants. Implementing informatic intervention within electronic medical records, when a disorder requiring special nutritional intervention is identified, will protect premature infants in this vulnerable setting. Standard of care management provided premature infants may be contraindicated in the context of a comorbid inborn error of metabolism.

Postnatal Pancraniosynostosis in a Patient With Infantile Hypophosphatasia.

Hypophosphatasia is a rare metabolic bone disorder that predisposes patients to craniosynostosis. Typically, patients born with hypophosphatasia will exhibit fused cranial sutures at birth. This is the first reported case of delayed onset of pancraniosynostosis in a patient with infantile hypophosphatasia. The severity of onset and delayed presentation in this patient are of interest and should give pause to those care providers who treat and evaluate patients with hypophosphatasia.

Abstract B25: Novel hepatic phenotypes caused by conditional c-Myc deletion

The transcription factor c-Myc (hereafter Myc) is among the most frequently deregulated oncoproteins. Inhibition of Myc triggers proliferative arrest of transformed cells and promotes tumor regression and/or apoptosis. Myc is also developmentally necessary and myc-/- embryos die at E9.5-10.5. However, Myc9s role in the maintenance of specific tissues has been shown to be of variable importance and necessity. For example, several studies of Myc9s role in promoting liver regeneration following partial hepatectomy (PH) have given conflicting results, although all show Myc to be generally dispensable for this function. We have used a conditional murine knockout (KO) model of Myc to study its role in liver regeneration. By employing a mycfl/fl;Alb-Cre+ model in which loss of Myc occurs perinatally, we studied non-oncogenic liver proliferation and metabolism in the absence of Myc signaling. We employed basic metabolic benchmarks of liver function including measurements of triglyceride levels, oxidative phosphorylation, and TCA cycle and electron transport chain function. At the molecular level, RNAseq was performed on isolated hepatocytes and the mitochondrial proteome was evaluated by both differential and unbiased mass spectrometry. At the time of active Myc excision, myc-/- mice had a significantly lower liver: body weight ratios relative to myc+/+ controls. However, this was reversed in older mice and was associated with the hepatic accumulation of neutral lipids, cholesterol and increased fatty acid β-oxidation in myc-/- mice. RNAseq on hepatocytes and Ingenuity Pathway analyses showed differences in 105 transcripts (q PH may provide an insufficiently sustained proliferative challenge to allow adequate evaluation of Myc9s potential role in liver regeneration. We therefore utilized a mouse model of hereditary tyrosinemia in which knockout of the gene encoding fumarylacetoacetate hydrolase (FAH) leads to hepatocellular death that can be rescued by the infusion of fah+/+ hepatocytes, which expand and eventually replace the fah-/- recipient hepatocytes. FAH-deficient animals could be rescued equally well by both myc+/+ and myc-/- hepatocytes. However, livers from the latter group showed excessive neutral lipid accumulation and fibrosis, reminiscent of non-alcoholic steatohepatitis (NASH). Taken together, our results provide unequivocal evidence that Myc is dispensable for long-term hepatic regeneration but is necessary to maintain proper lipid and steroid metabolism. In Myc9s absence the excessive accumulation of these intermediates predisposes to the development of a relatively mild pathology mimicking non-alcoholic fatty liver disease, which under the duress of chronic proliferation, progresses to a more severe NASH-like picture of end-stage liver disease. Our studies thus reveal a heretofore unappreciated role for Myc in hepatic metabolic homeostasis. Citation Format: Lia R. Edmunds, Lokendra Sharma, Peter Anthony Otero, Sonia D9Souza, James M. Dolezal, Xuemei Zeng, Ying Ding, Fei Ding, Megan E. Beck, Lisa E. Kratz, Jerry Vockley, Eric Goetzman, Donald Scott, Nathan Yates, Andrew W. Duncan, Edward V. Prochownik. Novel hepatic phenotypes caused by conditional c-Myc deletion. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B25.

Abstract 1259: c-Myc influences mitochondrial structure and function by regulating fusion and fission

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: c-Myc (Myc) is a global transcription factor that controls the expression of a large part of the human genome. The numerous Myc target genes function in a variety of normal cellular processes whose collective expression of these processes is referred to as the “Myc phenotype”. One such cellular process is the regulation of mitochondrial biogenesis. Objectives: To understand the specific influence that Myc has on mitochondrial structure and function. We hypothesize that Myc is involved in the regulation of both the production and destruction of mitochondria, and that the ability of Myc to maintain this balance has an impact on cellular transformation. Design/Method: Various cell lines that allow for the manipulation of Myc levels have been used. These include cells that have been engineered to have inducible Myc over-expression as well as inducible expression of Myc shRNA. These strains were used in a series of assays that measured different aspects of the Myc effect on mitochondrial structure and function. Most notably, metabolic pathways were measured by an extracellular flux analyzer and electron and confocal microscopy was performed to examine the structure of the mitochondria in detail. Results: The induction of Myc results in dramatic increases in overall mitochondrial mass, as well as increasing individual mitochondrial size and cristae density. This increase in recognizable mitochondria correlates to increases in oxidative phosphorylation and decreased mitochondrial loss through mitophagy. Finally, Myc levels correlate to increased mitochondrial inter-connectivity. This appears to be related to the ability of Myc to influence the expression of the mitochondrial fusion protein, Opa1 and the fission protein, DLP1. Conclusions Reached: The changes in mitochondrial structure and function are directly influenced by the expression of Myc. There appear to be multiple pathways involved in maintaining a specific mitochondrial mass, which emphasizes the importance of this organelle in cellular homeostasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1259. doi:10.1158/1538-7445.AM2011-1259