Bridging the Divide: Student Grand Rounds at the Interface of Basic Science and Clinical Medicine.

PROBLEM: As biomedical research and clinical medicine become increasingly complex, physician-scientists and clinically oriented biomedical researchers play important roles in bridging the gap between disciplines. A lack of educational programming that addresses the unique needs of students preparing for careers at the interface of basic science and clinical medicine may contribute to trainee attrition. APPROACH: The MD-PhD/LHB Grand Rounds was introduced in 2008 as a trainee-driven collaborative effort of the Harvard/Massachusetts Institute of Technology MD-PhD program at Harvard Medical School (HMS MD-PhD program), Harvard's Leder Human Biology and Translational Medicine (LHB) program, and the Brigham and Women's Hospital (BWH) Internal Medicine Department. Each of the program's approximately 4 sessions per year begins with dinner, followed by a clinical case presentation led by a BWH MD-PhD resident with a master clinician faculty discussant, then a research presentation by an LHB PhD student or an MD-PhD student on a basic science topic related to the clinical case, and time for socialization. OUTCOMES: In a July 2017 survey of participating students and residents, respondents reported being highly satisfied with the program. Mean satisfaction ratings were 4.3 (SD 0.5) for 12 MD-PhD students, 4.2 (SD 0.7) for 31 LHB students, and 4.4 (SD 0.9) for 5 residents on a 5-point scale (5 = very satisfied). Free-text responses suggested MD-PhD students valued opportunities for active engagement with the resident presenter and faculty discussant. LHB students appreciated the absence of medical jargon in the clinical presentations. Residents' reported reasons for participating included enjoyment of teaching and interaction with students. NEXT STEPS: The Harvard MD-PhD/LHB Grand Rounds can serve as a template for developing similar programs at other institutions. Research is needed to determine whether such grand rounds programs can help fix the leaky pipeline in the training of future physician-scientists and clinically oriented biomedical researchers.

Rapid immune reconstitution of SCID-X1 canines after G-CSF/AMD3100 mobilization and in vivo gene therapy.

Hematopoietic stem-cell gene therapy is a promising treatment of X-linked severe combined immunodeficiency disease (SCID-X1), but currently, it requires recipient conditioning, extensive cell manipulation, and sophisticated facilities. With these limitations in mind, we explored a simpler therapeutic approach to SCID-X1 treatment by direct IV administration of foamy virus (FV) vectors in the canine model. FV vectors were used because they have a favorable integration site profile and are resistant to serum inactivation. Here, we show improved efficacy of our in vivo gene therapy platform by mobilization with granulocyte colony-stimulating factor (G-CSF) and AMD3100 before injection of an optimized FV vector incorporating the human phosphoglycerate kinase enhancerless promoter. G-CSF/AMD3100 mobilization before FV vector delivery accelerated kinetics of CD3+ lymphocyte recovery, promoted thymopoiesis, and increased immune clonal diversity. Gene-corrected T lymphocytes exhibited a normal CD4:CD8 ratio and a broad T-cell receptor repertoire and showed restored γC-dependent signaling function. Treated animals showed normal primary and secondary antibody responses to bacteriophage immunization and evidence for immunoglobulin class switching. These results demonstrate safety and efficacy of an accessible, portable, and translatable platform with no conditioning regimen for the treatment of SCID-X1 and other genetic diseases.

(211)Astatine-Conjugated Monoclonal CD45 Antibody-Based Nonmyeloablative Conditioning for Stem Cell Gene Therapy.

Most hematopoietic stem cell gene therapy studies require host conditioning to allow for efficient engraftment of gene-modified cells. Conditioning regimens with lower treatment-related toxicities are especially relevant for the treatment of nonmalignant blood disorders, such as hemoglobinopathies and immunodeficiencies, and for patients who are otherwise ineligible for conventional high-dose conditioning. Radioimmunotherapy, which employs an α- or a ß-emitting radionuclide conjugated to a targeting antibody, is effective for delivering cytotoxic doses of radiation to a cell type of interest while minimizing off-target toxicity. Here, we demonstrate the feasibility of using a nonmyeloablative dose of a monoclonal anti-CD45 antibody conjugated to the α-emitter Astatine-211 ((211)At) to promote engraftment of an autologous gene-modified stem cell graft in the canine model. The doses used provided myelosuppression with rapid autologous recovery and minimal off-target toxicity. Engraftment levels were low in all dogs and reflected the low numbers of gene-modified cells infused. Our data suggest that a cell dose exceeding 1×10(6) cells/kg be used with nonmyeloablative doses of (211)At-anti-CD45 monoclonal antibodies for sustained engraftment in the dog model.

Gene therapy studies in a canine model of X-linked severe combined immunodeficiency.

Since the occurrence of T cell leukemias in the original human γ-retroviral gene therapy trials for X-linked severe combined immunodeficiency (XSCID), considerable effort has been devoted to developing safer vectors. This review summarizes gene therapy studies performed in a canine model of XSCID to evaluate the efficacy of γ-retroviral, lentiviral, and foamy viral vectors for treating XSCID and a novel method of vector delivery. These studies demonstrate that durable T cell reconstitution and thymopoiesis with no evidence of any serious adverse events and, in contrast to the human XSCID patients, sustained marking in myeloid cells and B cells with reconstitution of normal humoral immune function can be achieved for up to 5 years without any pretreatment conditioning. The presence of sustained levels of gene-marked T cells, B cells, and more importantly myeloid cells for almost 5 years is highly suggestive of transduction of either multipotent hematopoietic stem cells or very primitive committed progenitors.

Intravenous injection of a foamy virus vector to correct canine SCID-X1.

Current approaches to hematopoietic stem cell (HSC) gene therapy involve the collection and ex vivo manipulation of HSCs, a process associated with loss of stem cell multipotency and engraftment potential. An alternative approach for correcting blood-related diseases is the direct intravenous administration of viral vectors, so-called in vivo gene therapy. In this study, we evaluated the safety and efficacy of in vivo gene therapy using a foamy virus vector for the correction of canine X-linked severe combined immunodeficiency (SCID-X1). In newborn SCID-X1 dogs, injection of a foamy virus vector expressing the human IL2RG gene resulted in an expansion of lymphocytes expressing the common γ chain and the development of CD3(+) T lymphocytes. CD3(+) cells expressed CD4 and CD8 coreceptors, underwent antigen receptor gene rearrangement, and demonstrated functional maturity in response to T-cell mitogens. Retroviral integration site analysis in 4 animals revealed a polyclonal pattern of integration in all dogs with evidence for dominant clones. These results demonstrate that a foamy virus vector can be administered with therapeutic benefit in the SCID-X1 dog, a clinically relevant preclinical model for in vivo gene therapy.

Pigtailed macaques as a model to study long-term safety of lentivirus vector-mediated gene therapy for hemoglobinopathies.

Safely achieving long-term engraftment of genetically modified hematopoietic stem cells (HSCs) that maintain therapeutic transgene expression is the benchmark for successful application of gene therapy for hemoglobinopathies. We used the pigtailed macaque HSC transplantation model to ascertain the long-term safety and stability of a γ-globin lentivirus vector. We observed stable gene-modified cells and fetal hemoglobin expression for 3 years. Retrovirus integration site (RIS) analysis spanning 6 months to 3.1 years revealed vastly disparate integration profiles, and dynamic fluctuation of hematopoietic contribution from different gene-modified HSC clones without evidence for clonal dominance. There were no perturbations of the global gene-expression profile or expression of genes within a 300 kb region of RIS, including genes surrounding the most abundantly marked clones. Overall, a 3-year long follow-up revealed no evidence of genotoxicity of the γ-globin lentivirus vector with multilineage polyclonal hematopoiesis, and HSC clonal fluctuations that were not associated with transcriptome dysregulation.

A genomic analysis of chronological longevity factors in budding yeast.

Chronological life span (CLS) has been studied as an aging paradigm in yeast. A few conserved aging genes have been identified that modulate both chronological and replicative longevity in yeast as well as longevity in the nematode Caenorhabditis elegans; however, a comprehensive analysis of the relationship between genetic control of chronological longevity and aging in other model systems has yet to be reported. To address this question, we performed a functional genomic analysis of chronological longevity for 550 single-gene deletion strains, which accounts for approximately 12% of the viable homozygous diploid deletion strains in the yeast ORF deletion collection. This study identified 33 previously unknown determinants of CLS. We found no significant enrichment for enhanced CLS among deletions corresponding to yeast orthologs of worm aging genes or among replicatively long-lived deletion strains, although a trend toward overlap was noted. In contrast, a subset of gene deletions identified from a screen for reduced acidification of culture media during growth to stationary phase was enriched for increased CLS. These results suggest that genetic control of CLS under the most commonly utilized assay conditions does not strongly overlap with longevity determinants in C. elegans, with the existing confined to a small number of genetic pathways. These data also further support the model that acidification of the culture medium plays an important role in survival during chronological aging in synthetic medium, and suggest that chronological aging studies using alternate medium conditions may be more informative with regard to aging of multicellular eukaryotes.

Progeria syndromes and ageing: what is the connection?

One of the many debated topics in ageing research is whether progeroid syndromes are really accelerated forms of human ageing. The answer requires a better understanding of the normal ageing process and the molecular pathology underlying these rare diseases. Exciting recent findings regarding a severe human progeria, Hutchinson-Gilford progeria syndrome, have implicated molecular changes that are also linked to normal ageing, such as genome instability, telomere attrition, premature senescence and defective stem cell homeostasis in disease development. These observations, coupled with genetic studies of longevity, lead to a hypothesis whereby progeria syndromes accelerate a subset of the pathological changes that together drive the normal ageing process.

A genomic approach to yeast chronological aging.

Yeast is a useful model organism to study the genetic and biochemical mechanisms of aging. Genomic studies of aging in yeast have been limited, however, by traditional methodologies that require a large investment of labor and resources. In this chapter, we describe a newly-developed method for quantitatively measuring the chronological life span of each strain contained in the yeast ORF deletion collection. Our approach involves determining population survival by monitoring outgrowth kinetics using a Bioscreen C MBR shaker/incubator/plate reader. This method has accuracy comparable to traditional assays, while allowing for higher throughput and decreased variability in measurement.

A molecular mechanism of chronological aging in yeast.

The molecular mechanisms that cause organismal aging are a topic of intense scrutiny and debate. Dietary restriction extends the life span of many organisms, including yeast, and efforts are underway to understand the biochemical and genetic pathways that regulate this life span extension in model organisms. Here we describe the mechanism by which dietary restriction extends yeast chronological life span, defined as the length of time stationary yeast cells remain viable in a quiescent state. We find that aging under standard culture conditions is the result of a cell-extrinsic component that is linked to the pH of the culture medium. We identify acetic acid as a cell-extrinsic mediator of cell death during chronological aging, and demonstrate that dietary restriction, growth in a non-fermentable carbon source, or transferring cells to water increases chronological life span by reducing or eliminating extracellular acetic acid. Other life span extending environmental and genetic interventions, such as growth in high osmolarity media, deletion of SCH9 or RAS2, increase cellular resistance to acetic acid. We conclude that acetic acid induced mortality is the primary mechanism of chronological aging in yeast under standard conditions.

Suppression of proliferative defects associated with processing-defective lamin A mutants by hTERT or inactivation of p53.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, debilitating disease with early mortality and rapid onset of aging-associated pathologies. It is linked to mutations in LMNA, which encodes A-type nuclear lamins. The most frequent HGPS-associated LMNA mutation results in a protein, termed progerin, with an internal 50 amino acid deletion and, unlike normal A-type lamins, stable farnesylation. The cellular consequences of progerin expression underlying the HGPS phenotype remain poorly understood. Here, we stably expressed lamin A mutants, including progerin, in otherwise identical primary human fibroblasts to compare the effects of different mutants on nuclear morphology and cell proliferation. We find that expression of progerin leads to inhibition of proliferation in a high percentage of cells and slightly premature senescence in the population. Expression of a stably farnesylated mutant of lamin A phenocopied the immediate proliferative defects but did not result in premature senescence. Either p53 inhibition or, more surprisingly, expression of the catalytic subunit of telomerase (hTERT) suppressed the early proliferative defects associated with progerin expression. These findings lead us to propose that progerin may interfere with telomere structure or metabolism in a manner suppressible by increased telomerase levels and possibly link mechanisms leading to progeroid phenotypes to those of cell immortalization.

A method for high-throughput quantitative analysis of yeast chronological life span.

Chronological aging in yeast has been studied by maintaining cells in a quiescent-like stationary phase culture and monitoring cell survival over time. The composition of the growth medium can have a profound influence on chronological aging. For example, dietary restriction accomplished by lowering the glucose concentration of the medium significantly increases life span. Here we report a novel high-throughput method for measuring yeast chronological life span by monitoring outgrowth of aging cells using a Bioscreen C MBR machine. We show that this method provides survival data comparable to traditional methods, but with decreased variability. In addition to reducing the glucose concentration, we find that elevated amino acid levels or increased osmolarity of the growth medium is sufficient to increase chronological life span. We also report that life-span extension from dietary restriction does not require any of the five yeast sirtuins (Sir2, Hst1, Hst2, Hst3, or Hst4) either alone or in combination.

Naturally occurring IgM anti-leukocyte autoantibodies (IgM-ALA) inhibit T cell activation and chemotaxis.

The physiological relevance of naturally occurring IgM-ALA remains to be elucidated. These autoantibodies are present from birth and increase in diverse inflammatory states that are both infectious and noninfectious. Clinical observations showing significantly less acute allograft rejections in recipients having high IgM-ALA levels, led us to investigate whether IgM-ALA could have a functional role in attenuating T cell mediated inflammatory responses. In pursuit of this hypothesis, we did studies using IgM purified from the serum of normal individuals, patients with end stage renal disease, and HIV-1 infection. All preparations of IgM immunoprecipitated certain receptors e.g., CD3, CD4, CCR5, and CXCR4 from whole cell lysates but failed to immunoprecipitate IL-2R and HLA Ags. In physiological doses IgM down-regulated CD4, CD2 and CD86 but not CD8 and CD28, inhibited T cell proliferation, decreased production of certain proinflammatory cytokines e.g., TNF-alpha, IL-13 and IL-2, but not IFN- gamma, IL-1beta, GM-CSF, IL-6 and IL-8 and inhibited leukocyte chemotaxis. These inhibitory effects were more pronounced when using IgM from patients with high levels of IgM-ALA and these inhibitory effects were significantly reduced after using IgM preabsorbed with leukocytes. IgM-ALA binding to leukocytes was found to be highly specific, as <10% of IgM secreting B cell clones had IgM-ALA specificity with some clones having specificity for either T cells or monocytes. These findings support the concept that IgM-ALA provides an innate mechanism to regulate T cell mediated inflammatory responses.

Recent developments in yeast aging.

In the last decade, research into the molecular determinants of aging has progressed rapidly and much of this progress can be attributed to studies in invertebrate eukaryotic model organisms. Of these, single-celled yeast is the least complicated and most amenable to genetic and molecular manipulations. Supporting the use of this organism for aging research, increasing evidence has accumulated that a subset of pathways influencing longevity in yeast are conserved in other eukaryotes, including mammals. Here we briefly outline aging in yeast and describe recent findings that continue to keep this "simple" eukaryote at the forefront of aging research.

Correction of renal hypertension after kidney transplantation from a donor with Gitelman syndrome.

The relationship between the kidney and blood pressure control is complex. Monogenetic forms of hypertension have recently been identified that implicate specific mutations responsible for blood pressure control. The thiazide sensitive Na-Cl cotransporter (NCC) has been implicated in the control of blood pressure, however a direct link between the kidney NCC and blood pressure regulation is lacking. Here, we report a case of chimerism in which a kidney from a patient with Gitelman syndrome was transplanted into a non-Gitelman hypertensive recipient. After transplantation, postural hypotension resulted, necessitating discontinuation of all antihypertensive medications used for treatment of calcineurin-induced hypertension. This is the first reported case of acquired Gitelman syndrome after transplantation. Transplantation of a Gitelman "kidney" into a hypertensive recipient provides additional support for the role of the kidney NCC in blood pressure regulation. Furthermore, this case suggests the potential use of thiazide diuretics in the treatment of calcineurin-induced hypertension.