Male fertility preservation and restoration strategies for patients undergoing gonadotoxic therapies†.

Medical treatments for cancers or other conditions can lead to permanent infertility. Infertility is an insidious disease that impacts not only the ability to have a biological child but also the emotional well-being of the infertile individuals, relationships, finances, and overall health. Therefore, all patients should be educated about the effects of their medical treatments on future fertility and about fertility preservation options. The standard fertility preservation option for adolescent and adult men is sperm cryopreservation. Sperms can be frozen and stored for a long period, thawed at a later date, and used to achieve pregnancy with existing assisted reproductive technologies. However, sperm cryopreservation is not applicable for prepubertal patients who do not yet produce sperm. The only fertility preservation option available to prepubertal boys is testicular tissue cryopreservation. Next-generation technologies are being developed to mature those testicular cells or tissues to produce fertilization-competent sperms. When sperm and testicular tissues are not available for fertility preservation, inducing pluripotent stem cells derived from somatic cells, such as blood or skin, may provide an alternative path to produce sperms through a process call in vitro gametogenesis. This review describes standard and experimental options to preserve male fertility as well as the experimental options to produce functional spermatids or sperms from immature cryopreserved testicular tissues or somatic cells.

Induced pluripotent stem cell line from a mouse model of human azoospermia with a frameshift mutation Tex11_1260Ins(TT).

Infertility is a common disease that impacts 15% of reproductive age couples worldwide, and genetic causes are implicated in about half of those cases. Non-obstructive azoospermia is a severe form of male infertility that features spermatogenic failure resulting in no sperm in the ejaculate and severely reduces the chance to have biological children. We created a Tex11_1260Ins(TT) (1260GATA â†’ TTGGTA) mutant mouse that models the Tex11_1258(TT) mutation identified from a patient with nonobstructive azoospermia. The Tex11_1260Ins(TT) iPSC cells displayed characteristics of pluripotent-like morphology, expressed pluripotent protein markers, show normal karyotype, and can to differentiate into tissues of the three germ layers.

A Vietnamese human genetic variation database.

Large scale human genome projects have created tremendous human genome databases for some well-studied populations. Vietnam has about 95 million people (the 14th largest country by population in the world) of which more than 86% are Kinh people. To date, genetic studies for Vietnamese people mostly rely on genetic information from other populations. Building a Vietnamese human genetic variation database is a must for properly interpreting Vietnamese genetic variants. To this end, we sequenced 105 whole genomes and 200 whole exomes of 305 unrelated Kinh Vietnamese (KHV) people. We also included 101 other previously published KHV genomes to build a Vietnamese human genetic variation database of 406 KHV people. The KHV database contains 24.81 million variants (22.47 million single nucleotide polymorphisms (SNPs) and 2.34 million indels) of which 0.71 million variants are novel. It includes more than 99.3% of variants with a frequency of >1% in the KHV population. Noticeably, the KHV database revealed 107 variants reported in the human genome mutation database as pathological mutations with a frequency above 1% in the KHV population. The KHV database (available at https://genomes.vn) would be beneficial for genetic studies and medical applications not only for the Vietnamese population but also for other closely related populations.

UV-A1 therapy for nephrogenic systemic fibrosis.

BACKGROUND: Nephrogenic systemic fibrosis (NSF) is a rare sclerosing skin condition associated with end-stage renal disease and gadolinium exposure. Therapy for NSF is challenging, with few options other than preventing exposure to gadolinium and improving renal function through transplant. However, in some cases neither of these options is tenable. We report the successful use of UV-A1 phototherapy in 4 patients with NSF. OBSERVATIONS: Four patients with NSF were treated with UV-A1 phototherapy at a tertiary medical center from 2005 through 2007. To our knowledge, it is unique to this series that all patients were receiving hemodialysis before, during, and after therapy with UV-A1. All experienced improvement in the degree of induration, and 2 experienced improvement in mobility of the hands and legs. Total treatments ranged from 22 treatments (with a cumulative dose of 1855 J/cm(2)) to 50 treatments (total UV-A1 exposure, 3850 J/cm(2)). No adverse events were observed. CONCLUSIONS: Although no patient had complete resolution of indurated plaques, the improvement was substantial. For 2 patients, it resulted in a resumption of hand and leg mobility. As a result, UV-A1 therapy may represent a treatment for NSF when kidney transplantation is not an option or is delayed. Limitations of this study include the lack of a controlled trial, lack of quantification of gadolinium levels within tissue, and the lack of a defined grading scale for NSF severity.

Biopsy of the pigmented lesion--when and how.

The biopsy lies at the heart of the management of the suspected melanocytic neoplasm. Dermatologists are the ideal physicians to examine patients with suspect melanocytic lesions and an understanding of when and how to perform a biopsy is vital. Various algorithms have been formulated to allow for facilitation of the clinical examination, including the ABCDE rule, the Glasgow 7-point checklist, and the "ugly duckling" sign. Along with this, dermoscopy can increase the sensitivity of diagnosis. Proper training regarding dermatoscopy is essential, especially with algorithms such as the Menzies method, the 7-point checklist, and pattern analysis. Digital photography and digital dermatoscopy allows for surveillance of suspect nevi or patients with multiple nevi. For neoplasms suspected of being melanoma, an excision for diagnosis with 1- to 3-mm borders is ideal, although a shave, punch, or other incisional biopsy can be performed in special circumstances. Finally, research has allowed for promising technologies including gene profiling of tape-stripped samples along with automated software analysis of digital dermatoscopic images.

Cell surface restriction of EGFR by a tenascin cytotactin-encoded EGF-like repeat is preferential for motility-related signaling.

The 14th EGFL-repeat (Ten14) of human tenascin cytotactin activates the epidermal growth factor receptor (EGFR) with micromolar affinity; however, unlike EGF, Ten14-mediated activation of EGFR does not lead to receptor internalization. As the divergent signaling pathways downstream of EGFR have been shown to be triggered from plasma membrane and cytosolic locales, we investigated whether Ten14-mediated surface restriction of EGFR resulted in altered biochemical and cellular responses as compared to EGF. Molecules associated with migratory cascades were activated to a relatively greater extent in response to Ten14, with very weak activation of proliferation-associated cascades. Activation of phospholipase C gamma (PLCgamma) and m-calpain, associated with lamellipod protrusion and tail retraction, respectively, were noted at even at sub-saturating doses of Ten14. However, activation of ERK/MAPK, p90RSK, and Elk1, factors affecting proliferation, remained low even at high Ten14 concentrations. Similar activation profiles were observed for EGF-treated cells at 4 degrees C, a maneuver that limits receptor internalization. We demonstrate a concurrent effect of such altered signaling on biophysical responses-sustained migration was observed at levels of Ten14 that activated PLCgamma, but did not stimulate proliferation significantly. Here, we present a novel class of EGFR ligands that can potentially signal as a part of the extracellular matrix, triggering specific intracellular signaling cascades leading to a directed cellular response from an otherwise pleiotropic receptor. This work extends the signaling paradigm of EGFL repeat being presented in a restricted fashion as part of the extracellular matrix.

Tenascin cytotactin epidermal growth factor-like repeat binds epidermal growth factor receptor with low affinity.

Select epidermal growth factor (EGF)-like (EGFL) repeats of human tenascin cytotactin (tenascin C) can stimulate EGF receptor (EGFR) signaling, but activation requires micromolar concentrations of soluble EGFL repeats in contrast to subnanomolar concentrations of classical growth factors such as EGF. Using in silico homology modeling techniques, we generated a structure for one such repeat, the 14th EGFL repeat (Ten14). Ten14 assumes a tight EGF-like fold with truncated loops, consistent with circular dichroism studies. We generated bound structures for Ten14 with EGFR using two different approaches, resulting in two distinctly different conformations. Normal mode analysis of both structures indicated that the binding pocket of EGFR exhibits a significantly higher mobility in Ten14-EGFR complex compared to that of the EGF-EGFR complex; we hypothesized this may be attributed to loss of key high-affinity interactions within the Ten14-EGFR complex. We proved the efficacy of our in silico models by in vitro experiments. Surface plasmon resonance measurements yielded equilibrium constant K(D) of 74 microM for Ten14, approximately three orders of magnitude weaker than that of EGF. In accordance with our predicted bound models, Ten14 in monomeric form does not bind EGFR with sufficient stability so as to induce degradation of receptor, or undergo EGFR-mediated internalization over either the short (20 min) or long (48 h) term. This transient interaction with the receptor on the cell surface is in marked contrast to other EGFR ligands which cause EGFR transit through, and signaling from intracellular locales in addition to cell surface signaling.

Matrikines and matricryptins: Implications for cutaneous cancers and skin repair.

Dermatologists are faced daily with the need to optimize skin repair and excise cutaneous cancers. The extracellular matrix plays a pivotal role in cellular migration, proliferation, and gene regulation during wound healing and progression of melanoma, basal cell carcinoma, and squamous cell carcinoma. Within the last few years, a new class of ligand, the matrikine or matricryptin, has been characterized as subdomains of various ECM proteins capable of signaling to the cell through receptors, such as growth factor receptors. Two classes exist: the "natural" matrikines, which signal directly from the extracellular milieu and "cryptic" matrikines (matricryptins) that require proteolytic processing to reveal the ligand or to release the ligand from its ECM protein. Unlike traditional soluble growth factors, most matrikines possess low binding affinity to their receptors and are often presented in multiple valency that likely increase avidity to receptors. The presentation of these ligands within the ECM can result in unique outcomes. The EGF-like repeats of tenascin-C and laminin-5 signal to EGFR preferentially to upregulate migration during skin repair and tumor progression. Other matrikines in collagen, elastin, decorin, and laminin-1 can promote chemotaxis, mitogenesis, and metastasis in cancers, such as melanoma. Finally, the unique properties of matrikines have been utilized in cancer therapeutics and tissue engineering. Within the next few years, the nature and function of this emerging class of matrikine ligands will have an impact on dermatology, as these proteins are altered in wound repair and skin diseases.

Gabapentin withdrawal syndrome in the presence of a taper.

OBJECTIVE: To report a case report of a geriatric patient with a 5-year history of gabapentin use for enhanced bipolar control, who was tapered off of gabapentin over 1 week. The patient displayed unique withdrawal symptoms after the taper of gabapentin. METHODS: The patient is an 81-year-old white female with a life-long history of schizoaffective disorder with bipolar type I tendencies who had been prescribed gabapentin for 5 years. RESULTS: The patient displayed moderate upper respiratory tract infection symptoms and somatic complaints 1 day after termination of gabapentin. These symptoms gradually worsened until 10 days after, at which time she acutely developed severe mental status changes, severe somatic chest pain, and hypertension. Physical examination, electrolytes, electrocardiogram, computerized tomography, magnetic resonance imaging, and magnetic resonance angiography were all normal. Upon reintroduction of gabapentin, the patient returned to baseline within 1-2 days. CONCLUSIONS: Gabapentin is widely utilized currently for the chronic treatment of recalcitrant migraines, bipolar illness, pain, and epilepsy. It has a wide therapeutic index with few side effects and drug interactions, is not hepatically metabolized, and is excreted by the kidneys. Past reports have suggested that some withdrawal symptoms can present after 1-2 days upon abrupt discontinuation of gabapentin after chronic use within young to middle-aged patients. These symptoms mimic that of alcohol and benzodiazepine withdrawal purportedly due to a similar mechanism of action. Unique to this case is that this geriatric patient developed debilitating withdrawal symptoms after a gradual, week-long taper of gabapentin along with flu-like symptoms. It is proposed herein that a gabapentin taper should follow a course similar to that of a benzodiazepine taper -- slowly and over a period of weeks to months.

Extracellular matrix signaling through growth factor receptors during wound healing.

Recently, extracellular matrix components have been shown to contain domains that can interact with and activate receptors with intrinsic tyrosine kinase activity. These receptor tyrosine kinases are strong mediators of the cell responses of proliferation, migration, differentiation, and dedifferentiation. However, an interesting question is raised as to why cells would present growth factor receptor ligands in such a manner, as the majority of growth factors are small, soluble, or only transiently tethered ligands. With the exception of the discoidin domain receptors that bind collagen, the other described domains interact with a receptor that binds ubiquitous soluble peptide growth factors, the epidermal growth factor receptor. Unlike traditional growth factors, these individual "matrikine" domains within tenascin-C, laminin, collagen, and decorin possess relatively low binding affinity (high nanomolar or micromolar) and are often presented in multiple valency. The presentation of ligands within the extracellular matrix in this fashion might allow for unique biochemical and physiological outcomes. This new class of "matrikine" ligand may be critical for wound healing, as the majority of known extracellular matrix components possessing matrikines play a strong role, or are presented uniquely, during skin repair. Tenascin-C expression, for instance, is uniquely regulated spatially and has been proposed to present pro-migratory tracks during skin repair through its epidermal growth factor-like repeats. The epidermal growth factor-like repeats of laminin-5 act as cryptic ligands revealed upon matrix metalloproteinase-2 degradation of the surrounding extracellular matrix. The deletion of the discoidin domain receptors 1 and 2 for collagen have negative consequences on the role of fibroblasts and epithelial cells for matrix metalloproteinase production, migration, proliferation, and extracellular matrix turnover. Finally, decorin can bind to, inhibit, and down-regulate epidermal growth factor receptor levels and signaling, suggesting a tonic role of the epidermal growth factor binding domain of decorin in the resolution of wound healing. We provide a model framework for further studies into this emerging class of signals.

Keloid fibroblast responsiveness to epidermal growth factor and activation of downstream intracellular signaling pathways.

Keloids, which overgrow the boundaries of the original injury, represent aberrations in the fundamental process of wound healing that include over-abundant cell in-migration, cell proliferation, and inflammation, as well as increased extracellular matrix synthesis and defective remodeling. To understand the key events that result in the formation of these abnormal scars would open new avenues for better understanding of excessive repair, and might provide new therapeutic options. We examined epidermal growth factor receptor (EGFR)-induced cell motility in keloid fibroblasts, as this receptor initiates cell migration during normal wound repair. We show that keloid fibroblasts respond to EGF-induced cell migration but the response is somewhat diminished compared to normal adult fibroblasts (approximately 30% reduced); the mitogenic response was similarly blunted (approximately 5% reduced). Keloid fibroblasts express near normal levels of EGFR (82%), but show a much more attenuated activation of EGFR itself and the motility-associated phospholipase C-gamma. This was reflected in part by rapid loss of EGFR upon exposure to EGF. Interestingly, while extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) activation was relatively robust in keloid fibroblasts, the downstream triggering of the motility-associated calpain activity was blunted. This was reflected by high cell-substratum adhesiveness in the keloid fibroblasts. Thus, the blunted migratory response to EGF noted in keloid fibroblasts appears due to limited activation of two important biochemical switches for cell motility.

Aging-related attenuation of EGF receptor signaling is mediated in part by increased protein tyrosine phosphatase activity.

As fibroblasts near senescence, their responsiveness to external signals diminishes. This well-documented phenomenon likely underlies physiological deterioration and limited tissue regeneration in aging individuals. Understanding the underlying molecular mechanisms would provide opportunities to ameliorate these situations. A key stimulus for human dermal fibroblasts are ligands for the epidermal growth factor receptor (EGFR). We have shown earlier that EGFR expression decreases by about half in near senescent fibroblasts (Shiraha et al., 2000, J. Biol. Chem. 275 (25), 19343-19351). However, as the cell responses are nearly absent near senescence, other aging-related signal attenuation changes must also occur. Herein, we show that EGFR signaling as determined by receptor autophosphorylation is diminished over 80%, with a corresponding decrease in the phosphorylation of the immediate postreceptor adaptor Shc. Interestingly, we found that this was due at least in part to increased dephosphorylation of EGFR. The global cell phosphotyrosine phosphatase activity increased some threefold in near senescent cells. An initial survey of EGFR-associated protein tyrosine phosphatases (PTPases) showed that SHP-1 (PTPIC, HCP, SHPTP-1) and PTPIB levels are increased in parallel in these cells. Concomitantly, we also discovered an increase in expression of receptor protein tyrosine phosphatase alpha (RPTPalpha). Last, inhibition of protein tyrosine phosphatases by sodium orthovanadate in near senescent cells resulted in increased EGFR phosphorylation. These data support a model in which, near senescence, dermal fibroblasts become resistant to EGFR-mediated stimuli by a combination of receptor downregulation and increased signal attenuation.