Stem Cell Growth and Differentiation in Organ Culture: New Insights for Uterine Fibroid Treatment.

Organ culture allows for the understanding of normal and tumor cell biology, and tissues generally remain viable for 5-7 days. Strikingly, we determined that myometrial and MED12 mutant leiomyoma cells repopulated cell-depleted tissue slices after 20 days of culture. Using immunofluorescence and quantitative PCR of stem cell and undifferentiated cell markers, we observed clusters of CD49b+ cells in tumor slices. CD49b+ cells, however, were sparsely detected in the myometrial slices. Almost all LM cells strongly expressed Ki67, while only a few myometrial cells were stained for this proliferation marker. The CD73 marker was expressed only in tumor cells, whereas the mesenchymal stem cell receptor KIT was detected only in normal cells. HMGA2 and CD24 showed broader expression patterns and higher signal intensity in leiomyoma than in myometrial cells. In this study, we propose that activating CD49b+ stem cells in myometrium leads to asymmetrical division, giving rise to transit-amplifying KIT+ cells that differentiate to smooth muscle cells. On the contrary, activated leiomyoma CD49b+ cells symmetrically divide to form clusters of stem cells that divide and differentiate to smooth muscle cells without losing proliferation ability. In conclusion, normal and mutant stem cells can proliferate and differentiate in long-term organ culture, constituting a helpful platform for novel therapeutic discovery.

A high-throughput open-array qPCR gene panel to identify housekeeping genes suitable for myometrium and leiomyoma expression analysis.

OBJECTIVE: To evaluate 51 different housekeeping genes for their use as internal standards in myometrial and matched leiomyoma samples in proliferative and secretory phases. METHODS: RNA from 6 myometrium and matched leiomyoma samples was obtained from pre-menopausal women who underwent hysterectomy. Reverse-transcription and real-time quantitative PCR were achieved using TaqMan high-density open-array human endogenous control panel. RESULTS: Expression stability of 51 candidate genes was determined by GeNorm and NormFinder softwares. We identified 10 housekeeping genes, ARF1, MRPL19, FBXW2, PUM1, UBE2D2, EIF2B1, HPRT1, GUSB, ALAS1, and TRIM27, as the best set of normalization genes for comparing relative expression between leiomyoma and myometrium samples in proliferative and secretory phases. CONCLUSIONS: Adequate reference genes for accurate normalization are essential to compare gene expression between leiomyoma and myometrial samples. Ideal housekeeping genes must have stable expression patterns regardless of the sample type and menstrual cycle phase. In this study, we propose a set of 10 candidate genes with greater expression stability than those housekeeping genes commonly used in leiomyoma and myometrium tissues. Their use will improve the sensitivity and specificity of the gene expression analysis in these tissues.

Differentially regulated expression of neurokinin B (NKB)/NK3 receptor system in uterine leiomyomata.

STUDY QUESTION: Are the vasoactive peptide neurokinin B (NKB) and its preferred NK3 receptor (NK3R) differentially expressed in leiomyomas compared with normal myometrium? SUMMARY ANSWER: In leiomyomas, NKB is up-regulated and delocalized, while its preferred NK3R is also differentially regulated. WHAT IS KNOWN ALREADY: The expression of NKB/NK3R in the central nervous system is essential for proper function of the human reproductive axis. Additionally, this system is also widely expressed throughout the female genital tract. Leiomyomas impair fertility and are a major source of abnormal uterine bleeding. The aberrant synthesis of local factors can contribute to the pathological symptoms observed in women with leiomyomata. NKB could be one of these factors, since a vasoactive role of this peptide at a peripheral level has been observed in different systems and species, including humans. NK3R is strongly regulated by estrogens and its activation leads to nuclear translocation affecting chromatin structure and gene expression. STUDY DESIGN, SIZE, DURATION: Samples were obtained between 2006 and 2012 from 28 women of reproductive age at different stages of the menstrual cycle by hysterectomy. Leiomyomas and matched macroscopically normal myometrium from each woman were analysed in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: RT-PCR, quantitative real time, immunohistochemistry and in situ hybridization were used to investigate the pattern of expression of NKB/NK3R in tissue samples. MAIN RESULTS AND THE ROLE OF CHANCE: Expression of the gene encoding NKB (TAC3) was up-regulated 20-fold in leiomyomas, compared with matched myometrium (P = 0.0008). In tumour tissue, not only connective cells, but also myometrial, endothelial and vascular smooth muscle cells express TAC3 mRNA. Immunoreactivity to NKB was preferentially located in the smooth muscle cell nuclei from normal myometrium in the secretory phase, unlike matched leiomyoma, which showed a predominant cytoplasmic expression pattern. In the normal myometrium, TACR3 mRNA showed variable expression throughout the menstrual phases, with samples showing strong, reduced or no amplification. In leiomyoma, TACR3 was significantly up-regulated compared with matched myometrium (P = 0.0349). LIMITATIONS, REASONS FOR CAUTION: This study is descriptive and although we observed clear differential regulation of the NKB/NK3R system at mRNA and immunohistochemical staining levels in leiomyoma, future functional studies are needed to determine the precise role of NKB in the myometrium in normal and pathological conditions. In addition, further analysis (e.g. in cell culture models) will be required to determine the role of NKB in the nucleus of normal smooth muscle cells, whether nuclear translocation is mediated by NK3R and the consequences of the cytoplasmic expression of NKB in tumour cells. WIDER IMPLICATIONS OF THE FINDINGS: The NKB/NK3R system dysregulation observed in leiomyoma may contribute to the pathological symptoms observed in women with leiomyomata.

Neurotensin and neurotensin receptor 1 expression in human myometrium and uterine leiomyomas.

Leiomyomas or fibroids are the most frequently diagnosed tumors of the female genital tract, and their growth seems to be steroid-hormone dependent by a yet undetermined cellular and molecular mechanism. Sexual hormones induce the secretion of growth factor peptides and the expression of their receptors, stimulating cell proliferation. One of these factors is neurotensin, and increasing evidence suggests that it can promote growth of different cancer cells. Since there are no data on neurotensin expression in normal and tumoral uterine tissue, we have analyzed the expression of NTS and NTSR1 receptor using immunohistochemistry for protein detection, in situ hybridization to detect cells expressing NTS mRNA, and RT-PCR to detect NTSR1 transcript as well as any of the alternative splice variants recently described for this receptor. We found that NTS and NTSR1 are expressed in connective cells of normal myometrium. In leiomyomas, immunoreactivity for NTS and NTSR1 receptor is colocalized in the smooth muscle cells that are also transcribing NTS. Women receiving high doses of steroids for in vitro fertilization showed tumor growth and increased immunoreactivity for neurotensin and NTSR1 receptor. Interestingly, alternative splice variants of NTSR1 receptor were detected only in tumoral tissue. These findings suggest a role of steroid hormones inducing neurotensin expression in leiomyoma smooth muscle cells. In these cells, NTS could act autocrinally through NTSR1 receptor, promoting their proliferation.

Low-dose 3 mg levobupivacaine plus 10 microg fentanyl selective spinal anesthesia for gynecological outpatient laparoscopy.

BACKGROUND: Lidocaine selective spinal anesthesia has been effective for short-duration gynecological outpatient laparoscopy. We compared the intraoperative effectiveness, anesthetic recovery times, and patient satisfaction after levobupivacaine-fentanyl versus lidocaine-fentanyl spinal anesthesia during short-duration gynecological laparoscopy. METHODS: In this double-blind study, 52 healthy women scheduled to undergo tubal sterilization were randomly assigned to receive either intrathecal 10 mg lidocaine 2% plus 10 microg fentanyl (Group I) or intrathecal 3 mg levobupivacaine 0.5% plus 10 microg fentanyl (Group II), each solution made to a total volume of 3 mL with sterile water. The following variables were monitored intraoperatively: anesthesia onset time, need for anesthesia-analgesia supplementation, depth of sedation, surgical conditions, and occurrence of hemodynamic events. After surgery, motor block, proprioception, vibration sense, light touch, and Romberg's test were performed to evaluate whether the patients could bypass the postanesthesia care unit and be allowed to walk by themselves. Sensory block level was determined at 5, 10, and 15 min after anesthetic injection, and then every 15 min until resolution was complete. A difference of 25 min in sensory block resolution time was considered clinically relevant. RESULTS: Onset time and intraoperative conditions were comparable in both groups. No patient required general anesthesia to complete surgery. All patients from both groups bypassed the postanesthesia care unit. Ambulation took place after 27 (18-45) min in Group I and 30 (18-56) min in Group II (P = 0.24). Complete regression of spinal anesthesia occurred after 93 (65-120) min in Group I and 105 (78-150) min in Group II (P = 0.019); however, no differences were observed in time for home discharge 185 (150-300) min in Group I and 188 (125-300) min in Group II (P = 0.62). Global patient satisfaction was comparable between both groups. CONCLUSIONS: Levobupivacaine 3 mg plus 10 microg fentanyl may be used as a suitable alternative to 10 mg lidocaine plus 10 microg fentanyl for spinal anesthesia of short duration. It achieved a clinically equivalent time for resolution of sensory block, similar intraoperative conditions, and comparable patient satisfaction..

[Wegener's granulomatosis of the colon. Case report and review of the literature]. / Granulomatosis de Wegener con afección del colón. Presentación de un caso y revisión de la literatura.

INTRODUCTION: Wegener's granulomatosis is not very common and it is associated with colonic affection is extremely rare. It was first described by Klinger, and by Wegener in 1936. It is a systemic clinico-pathologic entity to be mediated by immune mechanisms. The most common clinical manifestations are of respiratory, renal, and vascular origins. Diagnosis is histologic. OBJECTIVE: Presentation of a clinical case and literature revision. MATERIAL AND METHODS: The patient was a 46 year old male who vegan 3 years previously with symmetrical arthralgia in ankles and knees, cough, purulent expectoration, intermittend fever, occasional moderated hematoquezia, profuse diarrhea, and abdominal pain. The patient was subjected to laboratory and cabinet studies, by means of which it was established a diagnosis secondary colitis colonic mucosa. Definitive histopathologic study confirmed diagnosis of colonic Wegener's granulomatosis.

Enfermedad de Hirschprung en el adulto joven / Hirscgprung's disease in the young adult

Objetivo: Describir los resultados obtenidos en 5 pacientes con enfermedad de Hirschprung, diagnosticados y operados en la edad adulta. Sede: Servicios de Cirugía General y Proctología del Hospital de Especialidades del Centro Médico Nacional La Raza del IMSS en México, D.F. Diseño: Estudio retrospectivo, longitudinal, con descripción de casos. Resultados: Cinco pacientes jóvenes, 3 hombres y 2 mujeres, con edades de 18 a 33 años, media de 23, fueron diagnosticados, mediante estudio clínico, radiológico, manométrico e histopatológico, de ser portadores de la enfermedad de hirschprung. Los principales datos clínicos fueron: constipación crónica, desnutrición y empleo frecuente de laxantes. En todos se corroboró radiológicamente megacolon; manométricamente, en 3 de ellos no se obtuvo el reflejo de la defecación al insuflar el balón rectal con 200 ml de aire (respuesta normal a los 90 ml), y en todos se demostró, mediante biopsia rectal, ausencia total de plexos sub-mucosos (Meissner y Henle) y mientéricos (Auerbach). Todos fueron operados, en 3 se realizó la operación se Soave, en 1 esfinterectomía rectal y en el otro, resección del sigmoides y de la unión recto-sigmoidea, no hubo mortalidad. Conclusión: A un cuando la enfermedad de Hischprug es rara en el adulto, debe pensarse en ella cuando se trate de adultos jóvenes, con constipación crónica y dilatación del colon.