Faculty Bibliography

In visceral leishmaniasis (VL), it is well-known that a patient in clinical remission of VL remains immune to reinfection, which provides a rationale for the feasibility of a vaccine against this deadly disease. In earlier studies, observation of significant cellular responses in treated Leishmania patients as well as in hamsters against leishmanial antigens from different fractions led to its further proteomic characterization, wherein S-Adenosyl-L-Homocysteine Hydrolase (AdoHcy) was identified as Th1 stimulatory protein. The present study includes immunological characterization of this protein, its cellular responses (lymphoproliferation, NO production and cytokine responses) in treated Leishmania infected hamsters and patients as well as prophylactic efficacy against Leishmania challenge in hamsters and the immune responses generated thereof. Significantly higher cellular responses were noticed against recombinant L. donovani S-Adenosyl-L-Homocysteine Hydrolase (rLdAdoHcy) as compared to soluble L. donovani antigen in treated samples. Moreover, stimulation of peripheral blood mononuclear cells with rLdAdoHcy up-regulated the levels of IFN-gamma, IL-12 and down-regulated IL-10. Furthermore, vaccination with rLdAdoHcy generated perceptible delayed type hypersensitivity response and exerted considerably good prophylactic efficacy ( approximately 70% inhibition) against L. donovani challenge. The efficacy was confirmed by the increased expression levels of inducible NO synthase and Th1-type cytokines, IFN-gamma and IL-12 and down-regulation of IL-4, IL-10 and TGF-beta. The results indicate towards the potentiality of rLdAdoHcy protein as a suitable vaccine candidate against VL

Stem cells are classified into embryonic stem cells and adult stem cells. An evolving alternative to conventional stem cell therapies is induced pluripotent stem cells (iPSCs), which have a multi-lineage potential comparable to conventionally acquired embryonic stem cells with the additional benefits of being less immunoreactive and avoiding many of the ethical concerns raised with the use of embryonic material. The ability to generate iPSCs from somatic cells provides tremendous promise for regenerative medicine. The breakthrough of iPSCs has raised the possibility that patient-specific iPSCs can provide autologous cells for cell therapy without the concern for immune rejection. iPSCs are also relevant tools for modeling human diseases and drugs screening. However, there are still several hurdles to overcome before iPSCs can be used for translational purposes. Here, we review the recent advances in somatic reprogramming and the challenges that must be overcome to move this strategy closer to clinical application.

KCNQ (voltage-gated K(+) channel family 7 (Kv7)) channels control cellular excitability and underlie the K(+) current sensitive to muscarinic receptor signaling (the M current) in sympathetic neurons. Here we show that the novel anti-epileptic drug retigabine (RTG) modulates channel function of pore-only modules (PMs) of the human Kv7.2 and Kv7.3 homomeric channels and of Kv7.2/3 heteromeric channels by prolonging the residence time in the open state. In addition, the Kv7 channel PMs are shown to recapitulate the single-channel permeation and pharmacological specificity characteristics of the corresponding full-length proteins in their native cellular context. A mutation (W265L) in the reconstituted Kv7.3 PM renders the channel insensitive to RTG and favors the conductive conformation of the PM, in agreement to what is observed when the Kv7.3 mutant is heterologously expressed. On the basis of the new findings and homology models of the closed and open conformations of the Kv7.3 PM, we propose a structural mechanism for the gating of the Kv7.3 PM and for the site of action of RTG as a Kv7.2/Kv7.3 K(+) current activator. The results validate the modular design of human Kv channels and highlight the PM as a high-fidelity target for drug screening of Kv channels.

The invention of new mouse molecular genetics techniques, initiated in the 1980s, has repeatedly expanded our ability to tackle exciting developmental biology problems. The brain is the most complex organ, and as such the more sophisticated the molecular genetics technique, the more impact they have on uncovering new insights into how our brain functions. I provide a general time line for the introduction of new techniques over the past 30 years and give examples of new discoveries in the neural development field that emanated from them. I include a look to what the future holds and argue that we are at the dawn of a very exciting age for young scientists interested in studying how the nervous system is constructed and functions with such precision.

The eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation-dependent integrated stress response (ISR), a component of the unfolded protein response, has long been known to regulate intermediary metabolism, but the details are poorly worked out. We report that profiling of mRNAs of transgenic mice harboring a ligand-activated skeletal muscle-specific derivative of the eIF2alpha protein kinase R-like ER kinase revealed the expected up-regulation of genes involved in amino acid biosynthesis and transport but also uncovered the induced expression and secretion of a myokine, fibroblast growth factor 21 (FGF21), that stimulates energy consumption and prevents obesity. The link between the ISR and FGF21 expression was further reinforced by the identification of a small-molecule ISR activator that promoted Fgf21 expression in cell-based screens and by implication of the ISR-inducible activating transcription factor 4 in the process. Our findings establish that eIF2alpha phosphorylation regulates not only cell-autonomous proteostasis and amino acid metabolism, but also affects non-cell-autonomous metabolic regulation by induced expression of a potent myokine.-Miyake, M., Nomura, A., Ogura, A., Takehana, K., Kitahara, Y., Takahara, K., Tsugawa, K., Miyamoto, C., Miura, N., Sato, R., Kurahashi, K., Harding, H. P., Oyadomari, M., Ron, D., Oyadomari, S. Skeletal muscle-specific eukaryotic translation initiation factor 2alpha phosphorylation controls amino acid metabolism and fibroblast growth factor 21-mediated non-cell-autonomous energy metabolism.

BACKGROUND: Previous studies have characterized differences in vitiligo associated with halo nevi, but the features of vitiligo presenting with halo nevus in children have yet to be fully described. AIMS: We sought to provide an epidemiologic and clinical comparison of cases of childhood vitiligo presenting with or without associated halo nevi. MATERIALS AND METHODS: This was a retrospective chart review of children diagnosed with vitiligo in an academic pediatric dermatology practice from January 1990 to November 2014. The characteristics of children with vitiligo with or without associated halo nevi were compared. RESULTS: Halo nevi were identified in 55 (26%) of 208 children with vitiligo. Patients with halo nevi were significantly more likely to be male and develop vitiligo at a later age. Children with vitiligo associated with halo nevi were more likely to present with generalized vitiligo, defined according to the presence of bilateral macules. DISCUSSION: There was no significant association between groups in the percentage of body surface area with vitiligo or family history of vitiligo or autoimmune diseases. Patients with halo nevi were no more likely to develop new areas of vitiligo during the follow-up period, but there was a nonsignificant trend toward a higher rate of repigmentation in vitiligo associated with halo nevus. CONCLUSION: Halo nevi are a common finding in children with vitiligo. The presence of a halo nevus in a child with vitiligo is associated with generalized vitiligo. The presence of a halo nevus does not significantly alter the risk of disease progression and rate of treatment.

OBJECTIVE: Understanding the mechanisms regulating normal and pathological angiogenesis is of great scientific and clinical interest. In this report, we show that mutations in 2 different aminoacyl-transfer RNA synthetases, threonyl tRNA synthetase (tarsy58) or isoleucyl tRNA synthetase (iarsy68), lead to similar increased branching angiogenesis in developing zebrafish. APPROACH AND RESULTS: The unfolded protein response pathway is activated by aminoacyl-transfer RNA synthetase deficiencies, and we show that unfolded protein response genes atf4, atf6, and xbp1, as well as the key proangiogenic ligand vascular endothelial growth factor (vegfaa), are all upregulated in tarsy58 and iarsy68 mutants. Finally, we show that the protein kinase RNA-like endoplasmic reticulum kinase-activating transcription factor 4 arm of the unfolded protein response pathway is necessary for both the elevated vegfaa levels and increased angiogenesis observed in tarsy58 mutants. CONCLUSIONS: Our results suggest that endoplasmic reticulum stress acts as a proangiogenic signal via unfolded protein response pathway-dependent upregulation of vegfaa.

The transcription factor hypoxia-inducible factor 1-alpha (HIF-1alpha) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1alpha is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1alpha degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1alpha as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFalpha, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 +/- 0.45 days vs. 19 +/- 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1alpha and upregulation of pro-angiogenic genes downstream of HIF-1alpha, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.

Navigation with fluorescence guidance has emerged in the last decade as a promising strategy to improve the efficacy of oncologic surgery. To achieve routine clinical use, the onus is on the surgical community to objectively assess the value of this technique. This assessment may facilitate both Food and Drug Administration approval of new optical imaging agents and reimbursement for the imaging procedures. It is critical to characterize fluorescence-guided procedural benefits over existing practices and to elucidate both the costs and the safety risks. This report is the result of a meeting of the International Society of Image Guided Surgery (www.isigs.org) on February 6, 2015, in Miami, Florida, and reflects a consensus of the participants' opinions. Our objective was to critically evaluate the imaging platform technology and optical imaging agents and to make recommendations for successful clinical trial development of this highly promising approach in oncologic surgery.