Therapeutic targeting of membrane-associated GRP78 in leukemia and lymphoma: preclinical efficacy in vitro and formal toxicity study of BMTP-78 in rodents and primates.

Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D(KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.

An AAVP-based solid-phase transducing matrix for transgene delivery: potential for translational applications.

A hybrid vector of adeno-associated virus and phage (termed AAVP) has been introduced as a platform for systemic ligand-directed delivery of transgenes to tumors over the past decade. A series of studies have evaluated the AAVP platform for potential theranostic or purely therapeutic applications in several tumor models. Sufficient ligand-directed tumor targeting consistently resulted in specific molecular-genetic imaging and/or anti-tumor responses to 'suicide' transgene delivery. However, efforts to optimize transduction efficiency are still ongoing. Here, we set out to expand the translational utility of AAVP by combining it with gold (Au) nanoparticles in order to generate a 'transducing matrix' for improved targeted gene delivery in solid phase. Targeted AAVP-based solid-phase transduction is superior to conventional transduction in soluble (aqueous) environments. This transducing matrix is stable and can be further modified with additional attributes (for example, magnetization) for targeted imaging and therapeutic gene delivery. Notably, it spontaneously assembles around cells in vitro to markedly enhance transduction capabilities compared with AAVP alone. This versatile nanoplatform may enable new applications of AAVP for transgene delivery in translational settings including, for example, efforts toward complex tissue patterning.

[Uterine subrogation: medical and legal aspects of the first legally supported case in Argentina]. / Subrogación uterina. Aspectos médicos y jurídicos del primer caso con sustento legal en la Argentina.

A woman aged 38 was referred to this center for surrogacy treatment, after subtotal ablation of her uterus due to a severe postpartum hemorrhage. Her hormonal profiles and ovarian structure were normal. The husband proved fertile and semen analysis was normal. The carrier, a woman 39 years old, fertile with two children of her own, and a long bonding friendship with the patient. After hormonal stimulation with gonadotropins and GnRH antagonist, six mature oocytes were obtained. These originated four embryos after in vitro fertilization, three of which were transferred to the carrier, achieving a singleton pregnancy which led to the birth of a normal child, now more than a year old. A lawsuit was filed after birth requesting the baby be registered with the biological parents name. The judge granted the request based on evidence and testimonies provided, international jurisprudence history and specification in Article 19 of the Argentine Constitution: "No inhabitant of the Nation shall be obliged to do what the law does not demand .nor be deprived of what it does not prohibit". This is an almost ideal example of the proceedings in a case of subrogation. However, we must always bear in mind the fact that in our country there is as yet no regulatory framework for these treatments, therefore there is a high probability of conflict.

Subrogación uterina: Aspectos médicos y jurídicos del primer caso con sustento legal en la Argentina / Uterine subrogation: Medical and legal aspects of the first legally supported case in Argentina

Una mujer de 38 años de edad fue derivada a nuestro centro para un tratamiento de maternidad subrogada, por haber sufrido la ablación subtotal de su útero a causa de una hemorragia puerperal grave. Su perfil hormonal y estructura ovárica eran normales. El esposo tenía fertilidad probada y un espermograma normal. La subrogante era una mujer de 39 años, fértil, con dos hijos propios y con un largo vínculo de amistad con la paciente. Luego de una estimulación con gonadotropinas y antagonista de Gn-RH se obtuvieron 6 ovocitos maduros por punción folicular, que originaron 4 embriones por fertilización in vitro. Tres de ellos evolucionaron y fueron transferidos a la subrogante, lográndose un embarazo único que concluyó con el nacimiento de una niña normal que ya tiene dos años cumplidos. Se inició una acción solicitando la inscripción de la beba ya nacida a nombre de los padres procreacionales. El juez finalmente la otorgó basándose en las pruebas y testimonios aportados, los antecedentes en la jurisprudencia internacional, y el hecho de que según el artículo 19 de la Constitución Nacional: "Ningún habitante de la Nación será obligado a hacer lo que no manda la ley, ni privado de lo que ella no prohíbe". Este es un ejemplo casi ideal de cómo se puede desarrollar un caso de subrogación. Sin embargo, hay que tener en cuenta que en nuestro país no existe aún un marco regulatorio para estos tratamientos, por lo que no queda exenta la posibilidad de conflicto.(AU)

A woman aged 38 was referred to this center for surrogacy treatment, after subtotal ablation of her uterus due to a severe postpartum hemorrhage. Her hormonal profiles and ovarian structure were normal. The husband proved fertile and semen analysis was normal. The carrier, a woman 39 years old, fertile with two children of her own, and a long bonding friendship with the patient. After hormonal stimulation with gonadotropins and GnRH antagonist, six mature oocytes were obtained. These originated four embryos after in vitro fertilization, three of which were transferred to the carrier, achieving a singleton pregnancy which led to the birth of a normal child, now more than a year old. A lawsuit was filed after birth requesting the baby be registered with the biological parents name. The judge granted the request based on evidence and testimonies provided, international jurisprudence history and specification in Article 19 of the Argentine Constitution: "No inhabitant of the Nation shall be obliged to do what the law does not demand .nor be deprived of what it does not prohibit". This is an almost ideal example of the proceedings in a case of subrogation .However, we must always bear in mind the fact that in our country there is as yet no regulatory framework for these treatments, therefore there is a high probability of conflict.(AU)

Subrogación uterina: Aspectos médicos y jurídicos del primer caso con sustento legal en la Argentina / Uterine subrogation: Medical and legal aspects of the first legally supported case in Argentina

Una mujer de 38 años de edad fue derivada a nuestro centro para un tratamiento de maternidad subrogada, por haber sufrido la ablación subtotal de su útero a causa de una hemorragia puerperal grave. Su perfil hormonal y estructura ovárica eran normales. El esposo tenía fertilidad probada y un espermograma normal. La subrogante era una mujer de 39 años, fértil, con dos hijos propios y con un largo vínculo de amistad con la paciente. Luego de una estimulación con gonadotropinas y antagonista de Gn-RH se obtuvieron 6 ovocitos maduros por punción folicular, que originaron 4 embriones por fertilización in vitro. Tres de ellos evolucionaron y fueron transferidos a la subrogante, lográndose un embarazo único que concluyó con el nacimiento de una niña normal que ya tiene dos años cumplidos. Se inició una acción solicitando la inscripción de la beba ya nacida a nombre de los padres procreacionales. El juez finalmente la otorgó basándose en las pruebas y testimonios aportados, los antecedentes en la jurisprudencia internacional, y el hecho de que según el artículo 19 de la Constitución Nacional: "Ningún habitante de la Nación será obligado a hacer lo que no manda la ley, ni privado de lo que ella no prohíbe". Este es un ejemplo casi ideal de cómo se puede desarrollar un caso de subrogación. Sin embargo, hay que tener en cuenta que en nuestro país no existe aún un marco regulatorio para estos tratamientos, por lo que no queda exenta la posibilidad de conflicto.

A woman aged 38 was referred to this center for surrogacy treatment, after subtotal ablation of her uterus due to a severe postpartum hemorrhage. Her hormonal profiles and ovarian structure were normal. The husband proved fertile and semen analysis was normal. The carrier, a woman 39 years old, fertile with two children of her own, and a long bonding friendship with the patient. After hormonal stimulation with gonadotropins and GnRH antagonist, six mature oocytes were obtained. These originated four embryos after in vitro fertilization, three of which were transferred to the carrier, achieving a singleton pregnancy which led to the birth of a normal child, now more than a year old. A lawsuit was filed after birth requesting the baby be registered with the biological parents name. The judge granted the request based on evidence and testimonies provided, international jurisprudence history and specification in Article 19 of the Argentine Constitution: "No inhabitant of the Nation shall be obliged to do what the law does not demand .nor be deprived of what it does not prohibit". This is an almost ideal example of the proceedings in a case of subrogation .However, we must always bear in mind the fact that in our country there is as yet no regulatory framework for these treatments, therefore there is a high probability of conflict.

Subrogación uterina. Aspectos médicos y jurídicos del primer caso con sustento legal en la Argentina. / [Uterine subrogation: medical and legal aspects of the first legally supported case in Argentina].

A woman aged 38 was referred to this center for surrogacy treatment, after subtotal ablation of her uterus due to a severe postpartum hemorrhage. Her hormonal profiles and ovarian structure were normal. The husband proved fertile and semen analysis was normal. The carrier, a woman 39 years old, fertile with two children of her own, and a long bonding friendship with the patient. After hormonal stimulation with gonadotropins and GnRH antagonist, six mature oocytes were obtained. These originated four embryos after in vitro fertilization, three of which were transferred to the carrier, achieving a singleton pregnancy which led to the birth of a normal child, now more than a year old. A lawsuit was filed after birth requesting the baby be registered with the biological parents name. The judge granted the request based on evidence and testimonies provided, international jurisprudence history and specification in Article 19 of the Argentine Constitution: "No inhabitant of the Nation shall be obliged to do what the law does not demand .nor be deprived of what it does not prohibit". This is an almost ideal example of the proceedings in a case of subrogation. However, we must always bear in mind the fact that in our country there is as yet no regulatory framework for these treatments, therefore there is a high probability of conflict.

Luminescent silica nanoparticles for cancer diagnosis.

Fluorescence imaging techniques are becoming essential for preclinical investigations, necessitating the development of suitable tools for in vivo measurements. Nanotechnology entered this field to help overcome many of the current technical limitations, and luminescent nanoparticles (NPs) are one of the most promising materials proposed for future diagnostic implementation. NPs also constitute a versatile platform that can allow facile multi-functionalization to perform multimodal imaging or theranostics (simultaneous diagnosis and therapy). In this contribution we have mainly focused on dye doped silica or silica-based NPs conjugated with targeting moieties to enable imaging of specific cancer cells. We also cite and briefly discuss a few non-targeted systems for completeness. We summarize common synthetic approaches to these materials, and then survey the most recent imaging applications of silica-based nanoparticles in cancer. The field of theranostics is particularly important and stimulating, so, even though it is not the central topic of this paper, we have included some significant examples. We conclude with a short section on NP-based systems already in clinical trials and examples of specific applications in childhood tumors. This review aims to describe and discuss, through focused examples, the great potential of these materials in the medical field, with the aim to encourage further research to implement applications, which today are still rare.

Blockade of inhibitors of apoptosis (IAPs) in combination with tumor-targeted delivery of tumor necrosis factor-α leads to synergistic antitumor activity.

In the current study, we examined whether the combination of tumor vasculature-targeted gene therapy with adeno-associated virus bacteriophage-tumor necrosis factor-α (AAVP-TNF-α) and/or the orally administered LCL161, an antagonist of inhibitors of apoptosis proteins (IAPs), enhanced antitumor efficacy without systemic toxicity. M21 human melanoma xenografts were grown subcutaneously in nude mice. Mice were treated according to one of four treatment regimens: AAVP-TNF-α alone (AAVP-TNF-α plus sodium acetate-acetic acid (NaAc) buffer) via tail vein injection; LCL161 alone (phosphate-buffered saline (PBS) plus LCL161) via oral gavage; AAVP-TNF-α plus LCL161; and PBS plus NaAc Buffer as a control group. Tumor volume, survival and toxicity were analyzed. AAVP trafficking and TNF-α production in vivo were detected on days 7 and 21 by real-time PCR, enzyme-linked immunosorbent assay and immunofluorescence. The levels of apoptosis and activation of caspases were assessed on days 7 and 21 by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) and immunofluorescence assays. Our results showed that the combination of AAVP-TNF-α and LCL161 significantly inhibited tumor growth and prolonged survival in mice with melanoma xenografts. The combination of AAVP-TNF-α and LCL161 was also significantly more effective than either agent alone, showing a synergistic effect without systemic toxicity.

Live birth of twins after IVF of oocytes that were cryopreserved almost 12 years before.

A 45-year-old woman received embryos from IVF by intracytoplasmic sperm injection (ICSI) with her own oocytes that were cryopreserved (slow freezing in a low-sodium medium) 11 years and 7 and a half months before, when she was 33 years old. From seven metaphase-II oocytes thawed, five survived, four were fertilized after ICSI and two cleaving embryos were transferred on day 3. A diamniotic dichorionic term pregnancy was achieved, ending with the delivery of two healthy girls. As far as is known, this case represents, to date, the longest storage period of cryopreserved human oocytes resulting in a live birth.

A 45-kDa ErbB3 secreted by prostate cancer cells promotes bone formation.

ErbB3 is a transmembrane growth factor receptor that has been implicated in the pathogenesis of human cancer. After finding that a truncated form of ErbB3 was present and upregulated in metastatic prostate cancer cells in lymph nodes and bone, we explored the pathophysiological functions of this unusual form of ErbB3 in the context of mouse calvaria as well as osteoblasts in vitro and the femur microenvironment in vivo. Here we demonstrate that prostate cancer cells expressed an alternatively spliced transcript that encodes a 45-kDa glycosylated protein (p45-sErbB3). The recombinant p45-sErbB3 purified from conditioned medium stimulated calvarial bone formation and induced osteoblast differentiation. Overexpression of p45-sErbB3 in the osteolytic prostate cancer cell line PC-3 converted its phenotype from bone lysing to bone forming upon injection into the femurs of immunodeficient mice. Further, we detected sErbB3 in plasma samples from patients with castration-resistant prostate cancer with bone metastasis. These observations establish that p45-sErbB3 is a structurally and functionally unique gene product of ErbB3 and suggest that p45-sErbB3 is likely one of the factors involved in the osteoblastic bone metastases of prostate cancer.

Optical systems for in vivo molecular imaging of cancer.

Progress toward a molecular characterization of cancer would have important clinical benefits; thus, there is an important need to image the molecular features of cancer in vivo. In this paper, we describe a comprehensive strategy to develop inexpensive, rugged and portable optical imaging systems for molecular imaging of cancer, which couples the development of optically active contrast agents with advances in functional genomics of cancer. We describe initial results obtained using optically active contrast agents to image the expression of three well known molecular signatures of neoplasia: including over expression of the epidermal growth factor receptor (EGFR), matrix metallo-proteases (MMPs), and oncoproteins associated with human papillomavirus (HPV) infection. At the same time, we are developing inexpensive, portable optical systems to image the morphologic and molecular signatures of neoplasia noninvasively in real time. These real-time, portable, inexpensive systems can provide tools to characterize the molecular features of cancer in vivo.

Birth resulting from transfer of blastocysts cryopreserved with propanediol after spontaneous hatching.

This paper reports the birth of a healthy baby resulting from transfer of blastocysts that were cryopreserved using propanediol after spontaneous hatching. A young infertile couple underwent IVF treatment in the clinic. After several IVF attempts, two births resulted; the first one with fresh embryos in 1996 after three IVF cycles, and the second one in 1999 (after a new IVF cycle in 1998) with frozen blastocysts that had remained cryopreserved in 1.5 mol/l propanediol and 0.1 mol/l sucrose after spontaneous hatching. This report of a healthy baby following transfer of hatched blastocysts frozen in propanediol supports further exploration of this approach.

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine.

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.

Clinical practice of embryo transfer.

In this review, several embryo transfer methods are considered, together with factors involved in achieving an effective transfer. The approach most used is transcervical intrauterine transfer. This is described in detail, together with the many variables influencing success, e.g. technical ability and training of personnel, catheter choice, value of a previous 'dummy transfer' and the need to minimize trauma during transfer and so prevent damage to the uterine lining, bleeding and uterine contractions. These factors can each negatively impact on pregnancy rates. Emphasis is put on quality, developmental stage and number of embryos to be transferred to limit multiple pregnancies and their unwanted side-effects. Culture to blastocyst stages and single embryo transfer when optimal quality embryos are available are discussed as means of avoiding multiple pregnancies. Reference is made to embryo cryopreservation and fertility following frozen embryo transfer. Other techniques, such as ultrasound-controlled transcervical intrauterine transfer, and ultrasound-controlled transmyometrial transfer, are reviewed. More invasive procedures, generically grouped as surgical embryo transfer, including gamete intra-Fallopian transfer (GIFT), zygote intra-Fallopian transfer (ZIFT), pronuclear stage transfer and embryo intra-Fallopian transfer (EIFT), are also described. These techniques had a place in IVF when the need to apply assisted reproductive techniques exceeded the capacity of most laboratories, but not today thanks to refined laboratory technology and improved understanding of implantation. Alternative assisted reproductive technologies, such as direct intra-follicular insemination (DIFI), Fallopian spermatic perfusion (FSP), peritoneal oocyte stage and sperm transfer and intra-vaginal culture (IVC), are mentioned briefly.

Birth of two babies using oocytes that were cryopreserved in a choline-based freezing medium.

BACKGROUND: Oocyte cryopreservation may have significant potential for assisted reproductive technology. However, to date, successful results have been limited. We report a preliminary series of IVF outcomes after fertilization of oocytes that were frozen in a low-sodium medium. METHODS: In this retrospective analysis, 12 patients (21-41 years old), who underwent IVF in a fertility clinic affiliated to the University of Buenos Aires, had oocytes cryopreserved in a modified phosphate buffered saline medium, in which sodium chloride was replaced by choline chloride. A slow-freezing, rapid-thawing protocol was used and oocytes were inseminated by ICSI. Outcome measures included oocyte survival, fertilization, implantation and pregnancy rates. RESULTS: Median oocyte survival was 63%. Median fertilization rate was 59%. Overall implantation rate was 25%. Six clinical pregnancies were achieved; two of these pregnancies went to term resulting in the birth of two babies. CONCLUSIONS: To the best of our knowledge, these are the first pregnancies and normal births using oocytes that were cryopreserved in a choline-based medium. The small sample size prevents us from concluding that freezing in a low-sodium medium is superior to using a conventional one.

Impact factor of medical journals: problems in geriatrics.

The Impact Factor (IF) is one of the most reliable methods for evaluating the quality of scientific journals. To date, no one has studied the IF of geriatric journals over time. Our aims were 1) to evaluate the IF trend of geriatric journals in general, and with respect to internal medicine, which is the nearest discipline, and 2) to assess the proportion of papers on common geriatric topics published in geriatric journals, with respect to other general periodicals. The current literature was studied to define what IF is and describe its good points and deficiencies. We then analyzed the situation regarding geriatric journals. A bibliometric analysis addressed the kind of journal interested in important geriatric themes (syncope, falls, hip fractures, delirium). Geriatric journals have a low IF, but their importance has been growing over the years. Despite this, papers regarding geriatric themes are published mainly in non-specific journals, and the number of publications regarding syncope, falls, hip fractures, and delirium has not increased during the period in question. In conclusion, it is difficult to use the IF for comparisons between different disciplines, in particular when the one under consideration (geriatrics and gerontology) is spread over a wide range of medical fields. The increase in IF of geriatric journals over time is the most important result of our analysis; this finding will encourage geriatric scientists to submit their papers to geriatric periodicals and will increase the specificity of the gerontological discipline.

Cysteamine supplementation during in vitro maturation and embryo culture: a useful tool for increasing the efficiency of bovine in vitro embryo production.

Cysteamine when added during in vitro maturation (IVM) or in vitro embryo culture (IVC) stimulates glutathione (GSH) synthesis and improves embryo developmental rates. This suggests that GSH synthesis is decreased in the in vitro produced embryo. The present study was carried out to evaluate if addition of cysteamine to culture medium at the same time, during IVM and IVC of bovine oocytes, may promote an overall improvement on the developmental rate and embryo quality. Oocytes were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and 0 or 100 microM of cysteamine for 24 hr. After IVM, the oocytes were fertilized (day 0). Day 2 embryos (2-8 cell) were washed and transferred to fresh IVC medium supplemented with 0, 25, 50, or 100 microM of cysteamine and cultured for 48 hr. After this, embryos were cultured in IVC medium without cysteamine until day 8 of IVC. In the present study, we confirmed our previous results by demonstrating that the percentage of embryos that developed to the blastocyst stage was significantly higher (P < 0.05) when 100 microM of cysteamine was added during IVM, and this was further improved when 100 and 50 microM of cysteamine where present during IVM and IVC, respectively (P < 0.05). After cryopreservation, no differences were observed on embryo development, but a significant increase on embryo hatching was found between unsupplemented and supplemented oocytes with 100 and 50 microM of cysteamine during IVM and IVC, respectively (P < 0.05). We can conclude that GSH synthesis stimulation during bovine IVM with cysteamine, concomitant with GSH stimulation during IVC, will be a useful and simple tool for increasing the efficiency of in vitro bovine embryo production.

Birth of a healthy baby after transfer of embryos that were cryopreserved for 8.9 years.

OBJECTIVE: To report successful pregnancy from embryos that had been cryopreserved for 8.9 years. DESIGN: Case report. SETTING: Fertility clinic of the University of Buenos Aires School of Medicine. PATIENT(S): A 43-year-old woman with ovarian failure and a 45-year-old man with nonobstructive azoospermia underwent embryo donation.I NTERVENTION(S): Five embryos that had been cryopreserved and stored since 1989 were thawed in 1998 and donated to an infertile couple. Endometrial preparation was performed with 17beta-estradiol and progesterone. The four embryos with better morphologic characteristics were transferred into the uterus. MAIN OUTCOME MEASURE(S): Post-thaw embryo survival, pregnancy, and birth. RESULT(S): Embryo survival was satisfactory as assessed by morphology. Pregnancy was confirmed by ultrasonography. A healthy baby weighing 2120 g was delivered by cesarean section at 36 weeks. CONCLUSION(S): Human 8-cell embryos may be viable after extended storage and can result in successful pregnancy.