Systemic injection of AAV9 carrying a periostin promoter targets gene expression to a myofibroblast-like lineage in mouse hearts after reperfused myocardial infarction.

Adeno-associated virus (AAV) has been used to direct gene transfer to a variety of tissues, including heart, liver, skeletal muscle, brain, kidney and lung, but it has not previously been shown to effectively target fibroblasts in vivo, including cardiac fibroblasts. We constructed expression cassettes using a modified periostin promoter to drive gene expression in a cardiac myofibroblast-like lineage, with only occasional spillover into cardiomyocyte-like cells. We compared AAV serotypes 6 and 9 and found robust gene expression when the vectors were delivered by systemic injection after myocardial infarction (MI), with little expression in healthy, non-infarcted mice. AAV9 provided expression in a greater number of cells than AAV6, with reporter gene expression visible in the cardiac infarct and border zones from 5 to 62 days post MI, as assessed by luciferase and Cre-activated green fluorescent protein expression. Although common myofibroblast markers were expressed in low abundance, most of the targeted cells expressed myosin IIb, an embryonic form of smooth muscle myosin heavy chain that has previously been associated with myofibroblasts after reperfused MI. This study is the first to demonstrate AAV-mediated expression in a potentially novel myofibroblast-like lineage in mouse hearts post MI and may open new avenues of gene therapy to treat patients surviving MI.

Intracerebroventricular delivery of self-complementary adeno-associated virus serotype 9 to the adult rat brain.

Gene therapy for the central nervous system is poised to become a powerful treatment for numerous neurological disorders. Adeno-associated viral vectors based on serotype 9 (AAV9) have proven themselves to be strong candidates for delivering gene-based therapies throughout the brain and spinal cord when administered intravenously, intrathecally, intracisternally, and intracerebroventricularly (i.c.v.). Previous studies of i.c.v.-delivered self-complimentary AAV9 have been performed in neonatal mice with delivery of a single dose. However, before clinical trials can be considered, more information is required about the dose-response relationship for transduction efficiency in adult animals. In the current study, three doses of self-complementary AAV9 were administered to adult rats. High levels of transduction were observed in the hippocampus, cerebellum and cerebral cortex, and transduction increased with increasing dosage. Both neurons and astrocytes were transduced. There was no evidence of astrocytosis at the doses tested. Preliminary results from pigs receiving i.c.v. self-complementary AAV9 are also presented. The results of this study will serve to inform dosing studies in large animal models before clinical testing.

Effects of static stretching on leg power during cycling.

AIM: This study investigated the effects of warm-up involving static stretching on leg power. It was expected that the inclusion of static stretching in the warm-up would decrease leg power. METHODS: Twenty-seven healthy volunteers (16 male, 11 female) participated in the study. A prospective, repeated measures design was implemented where volunteers underwent 2 testing sessions at least 24 hours apart. One testing session involved a control warm-up (5 min submaximal cycling) followed by 4 10-s leg power tests at 5, 20, 40 and 60 min postwarm-up. For the other testing session, the subjects performed 5 min of submaximal cycling followed by 15 min of lower body static stretching and then the four leg power tests. Relative peak power, time to peak power and relative total work were measured for each leg power test. RESULTS: Peak power and total work were significantly greater after the static stretching warm-up compared to the control warm-up on all power tests. Peak power was achieved more quickly for the static stretching warm-up compared to the control warm-up on the 5 min test only. CONCLUSIONS: A warm up that includes static stretching has beneficial effects on anaerobic power events in comparison to submaximal cycling alone.

Leptin expression in human mammary epithelial cells and breast milk.

Leptin has recently been shown to be produced by the human placenta and potentially plays a role in fetal and neonatal growth. Many functions of the placenta are replaced by the mammary gland in terms of providing critical growth factors for the newborn. In this study, we show that leptin is produced by human mammary epithelial cells as revealed by RT/PCR analysis of total RNA from mammary gland and immunohistochemical staining of breast tissue, cultured mammary epithelial cells, and secretory epithelial cells present in human milk. We also verify that immunoreactive leptin is present in whole milk at 30- to 150-fold higher concentrations than skim milk. We propose that leptin is secreted by mammary epithelial cells in milk fat globules, which partition into the lipid portion of breast milk.

Neonatal herpes simplex infection possibly acquired via maternal breast milk.

A newborn infant with disseminated herpes simplex virus type 1 (HSV-1) infection was determined serologically to have acquired the infection postnatally; his mother was found to have HSV-1 in her breast milk but had no history of genital lesions and negative viral cultures of cervix, vagina, and throat. We suggest that HSV-infected maternal breast milk may be a source of this infection for susceptible infants.

Technetium colloid bone marrow imaging in Fanconi's anemia.

Four patients with Fanconi's anemia were evaluated with 99mTc-sulfur colloid bone marrow scans. The scans revealed similar paradoxical and irregular tracer distribution in all four patients. Normal to increased activity was demonstrated in the proximal metaphyses of the humeri with varying degrees of increased activity in more primitive marrow sites in the distal diaphyses of the humeri and tibia, the distal metaphyses of the humeri and femora, and the proximal metaphyses of the ulnae, radii, and tibia. Skip areas of normal distribution were seen in the proximal diaphyses of the humeri and femora. Although the scan reflects only the reticuloendothelial portion of bone marrow, it may be of some value in the differential diagnosis of pancytopenia.

Vacuolated cytotrophoblast: a subpopulation of trophoblast in the chorion laeve.

In summary, immunocytochemical analysis of the placenta and fetal membranes discloses two subpopulations of trophoblastic cells in the chorion laeve with distinct morphologic and immunocytochemical features. Both subpopulations are composed of mononucleate cells but one contains clear vacuolated cytoplasm whereas the other contains non-vacuolated eosinophilic cytoplasm. The vacuolated cells are placental alkaline phosphatase (PLAP) positive and human placental lactogen (hPL) negative, whereas the eosinophilic cells are PLAP negative and hPL positive. Both cell types contained immunoreactive keratin and epithelial membrane antigen, but are negative for human chorionic gonadotropin(beta), pregnancy specific beta I-glycoprotein, and prolactin. Electron microscopic studies of the vacuolated cells show that these cells contain numerous lucent non-membrane bound lipid droplets and pinocytotic vesicles. In addition they contain numerous intracellular filaments and desmosomes corresponding to the immuno-cytochemical localization of keratin. Their precise function is not clear, but the abundance of PLAP, an enzyme associated with absorption, suggests that these cells may be involved with maternal fetal transport. The vacuolated cells appear to be limited to the chorion. Their characteristic morphologic and immunocytochemical features and unique anatomic distribution suggests that they represent a distinctive subpopulation of trophoblastic cells for which we propose the term 'vacuolated cytotrophoblast'.

Mitotically active leiomyomas of the uterus.

Seventy-three smooth-muscle tumors of the uterus, with five to nine mitotic figures per 10 high-power fields but lacking cytologic atypia, were studied. Pathologic features assessed included necrosis, hemorrhage, vascular intrusion, abnormal mitoses, and the number of mitotic figures. Vascular ingrowth by the tumor was present in 11 neoplasms. Follow-up ranged from 23 months to 15 years, with all patients alive and well at last contact. In 14 patients, myomectomy was the only treatment. Thirteen of these patients demonstrated no subsequent residual or recurrent tumor. One woman developed a persistent or recurrent mitotically active leiomyoma 8 years after myomectomy. We conclude that mitotically active smooth-muscle tumors of the uterus, having five to nine mitoses per 10 high-power fields and no cellular atypia, have a metastatic rate too low to be regarded as sarcomas. Hysterectomy need not automatically follow myomectomy if follow-up is done.

Congenital hemangiopericytoma: an unusual vascular neoplasm of infancy.

BACKGROUND: Hemangiopericytoma is an uncommon tumor of infants, which originates from the vascular pericytes. Although generally considered to benign, metastases can occur. METHODS: Five cases of congenital hemangiopericytoma were seen in infants; all were found in females. The mean age at diagnosis was 7 weeks (range, birth to 10 weeks). The lesions were located in the neck, the parotid, the axilla, and the retroperitoneum. One neck lesion was detected prenatally by ultrasonography. Each lesion was resected. The diagnosis of congenital hemangiopericytoma was established only after histologic examination. RESULTS: No evidence of recurrence has been found in four of the children. However, intrathoracic and intracranial metastases developed in one child with a neck lesion 28 months after the original resection, and the child was treated with chemotherapy, but she died of progressive disease. CONCLUSIONS: We advocate the consideration of congenital hemangiopericytoma in the newborn infant with a vascular mass. Because congenital hemangiopericytoma is unresponsive to steroid therapy, unlike other vascular malformations, resection is the treatment of choice. Long-term postoperative follow-up is essential for the early detection of metastases.

Intermediate trophoblast in uterine curettings in the diagnosis of ectopic pregnancy.

Confirming the presence of an intrauterine implantation site in endometrial curettings without recognizable chorionic villi is important in the diagnosis and management of ectopic pregnancy. In order to evaluate histologic features that might be useful in establishing the diagnosis of an intrauterine gestation, we conducted a retrospective review of 150 endometrial curettings obtained from women with incomplete abortions or ectopic pregnancies. The specimens were divided into three groups: 1) intrauterine gestations with villi, 2) intrauterine gestations without villi, and 3) known ectopic pregnancies. Differences in the histologic appearance of the endometrium between intrauterine and extrauterine pregnancies were evaluated, with emphasis on 34 curettings from intrauterine gestations in which no chorionic villi were identified and 12 curettings from ectopic gestations. Histologic features assessed in these 46 cases included the presence of intermediate trophoblastic cells; caliber, thickness, and hyalinization of spiral arteries; the presence of a fibrinoid matrix; the Arias-Stella phenomenon; decidua; inflammation; and thrombosis. Intermediate trophoblast in vessels and/or decidua was recognized by conventional light microscopy in 14 (41.2%) of 34 intrauterine cases with no villi, but was identified by immunoperoxidase reactions for hPL in 24 (70.6%) and keratin in 20 (58.8%) of these cases. Endometria from intrauterine gestations also demonstrated a significant increase in arterial wall thickness, hyalinization and lumen diameter, and thrombosis when compared with the endometria associated with ectopic pregnancies. Intermediate trophoblast, enlarged hyalinized spiral arteries, and fibrinoid matrix were not seen in any of the ectopic pregnancies. Based on these findings, intrauterine implantation can be diagnosed, in the absence of villi, by the presence of intermediate trophoblast associated with enlarged vessels replaced by hyaline, or with fragments of fibrinoid matrix.

Colposcopy in pregnancy: directed brush cytology compared with cervical biopsy.

OBJECTIVE: To evaluate colposcopically directed brush cytology as a substitute for directed biopsy of acetowhite lesions identified during pregnancy. METHODS: Pregnant patients eligible for the study were referred for colposcopic evaluation for either newly diagnosed abnormal cervical cytology or follow-up of a previously diagnosed squamous intraepithelial lesion (SIL). All patients with acetowhite lesions underwent colposcopically directed brush cytology followed by directed biopsy. RESULTS: Of 81 pregnant patients referred, 50 paired samples of colposcopically directed brush cytology and directed biopsies were evaluated from 49 patients. One patient was sampled in the first and third trimesters and one patient's brush cytology was unsatisfactory for interpretation because of clumping artifact, leaving 49 brush-biopsy pairs that were satisfactory for examination. One patient in the study group had an intrauterine fetal death of uncertain cause, remote from the time of biopsy. Compared with the corresponding biopsy, the directed brush caused significantly less blood loss (P < .001). For all diagnostic categories, directed cytology demonstrated a good degree of correlation with biopsy (kappa = 0.73). The brush technique correctly identified 12 of 14 cases (86%) of biopsy-proved cervical intraepithelial neoplasia II-III as high-grade SIL. If one considers "atypical squamous cells, favor human papillomavirus effect" as a true positive, brush sensitivity was 88 +/- 9% and specificity was 74 +/- 12%, with an accuracy of 80%. CONCLUSION: In the absence of lesions suspicious for carcinoma, colposcopically directed brush cytology is a safe substitute for directed biopsy in pregnant patients.

Characteristics of gonadotropin response, follicular development, and endometrial growth and maturation across consecutive cycles of clomiphene citrate treatment.

OBJECTIVE: To examine the patterns of gonadotropin response, follicular development, and endometrial growth and maturation across consecutive cycles of clomiphene citrate (CC) treatment. DESIGN: Prospective analysis of cycle characteristics. SETTING: Academic tertiary medical center. PATIENTS: Nineteen consenting anovulatory infertile women receiving standardized, cyclic, incremental treatment with CC (50 to 150 mg/d, cycle days 5 to 9) for ovulation induction. INTERVENTIONS: In each of up to six consecutive treatment cycles, urinary LH was monitored twice daily from cycle day 10 until detection of the LH surge or day 21; blood samples and transvaginal ultrasound (US) examination were obtained on cycle days 3, 10, and every 1 to 3 days thereafter until collapse of the dominant follicle. Endometrial biopsy was performed 11 to 13 days after the LH surge in the first, third, and sixth ovulatory cycle. RESULTS: Follicular phase duration, peak follicular diameter, the number of preovulatory follicles, and peak endometrial thickness and echo pattern remained consistent across consecutive ovulatory (n = 55) and anovulatory (n = 23) treatment cycles. Endometrial dating was > or = 3 days out of phase in 2 of 31 (6%) cycles sampled. Peak serum E2 and P concentrations did not vary with cycle number or correlate with endometrial thickness or echo pattern. Cycle day 10 FSH levels were significantly higher in ovulatory subjects than in anovulatory subjects. CONCLUSIONS: Patterns of gonadotropin response, follicular development, and endometrial growth and maturation remain consistent across consecutive cycles of CC treatment.

Management of ovarian neoplasms of low malignant potential.

Ovarian epithelial carcinomas of low malignant potential occur at an earlier age and in an earlier stage than invasive ovarian carcinomas. A thorough knowledge of the histologic characteristics and adequate sampling are vital to making a correct diagnosis. Surgical resection alone is the treatment for Stage I disease, which accounts for 66% of all patients with this disease. Treatment of later stages is controversial, but surgical resection alone may also be adequate therapy for most patients with a more advanced stage. There is a small subpopulation of patients who die with a tumor of low malignant potential that, for poorly understood reasons, progresses.

Managing mature cystic teratomas of the ovary.

Mature cystic teratomas (MCT), commonly called dermoid cysts, are the most common benign germ cell tumors of the ovary in women of reproductive age. Future fertility is of major concern among these women; therefore, the surgical management must focus on preserving ovarian tissue and minimizing adhesion formation. Patients requiring surgery should be appropriately counseled about the risks and benefits of laparoscopy and laparotomy, the risks of intraoperative MCT spillage and adhesion formation. In addition, the risks of recurrence and malignant transformation should be discussed. The parents of children with MCTs have the same concerns as older women and a similar discussion should take place. The goal of this article is to review these issues and provide the physician with the information to counsel their patients preoperatively.

Pediatric laryngotracheal reconstruction with cartilage grafts and endotracheal tube stenting: the single-stage approach.

Laryngotracheal reconstruction (LTR) comprises five stages: 1, characterization of the stenosis; 2, expansion of the lumen; 3, stabilization of the enlarged lumen framework; 4, healing of the surgical site; and 5, decannulation. Single-stage LTR (SS-LTR) combines and compresses stages 3 through 5 (stabilization, healing, and decannulation) into a brief period of postoperative intubation. At Children's Hospital Medical Center in Cincinnati, Ohio, from January 1987 to December 1993, 116 reconstructive procedures were performed by using postoperative intubation. Nine were tracheoplasty or stoma-revision procedures in isolation, leaving 107 SS-LTR procedures that included the laryngotracheal complex. The use of SS-LTR has increased to 30% of LTRs performed in 1993. Data are presented on the success of SS-LTR as measured by the number of decannulations and extubations achieved.

Restoring patency of thrombosed catheters with cryopreserved urokinase.

In order to eliminate wasting vials of urokinase, 5000 U/ml aliquots were reconstituted and frozen. The urokinase remained effective for clearing thrombosed catheters for as long as 9 months. When catheters become occluded, patency can be restored by previously frozen urokinase.

Magnetic resonance imaging determination of tracheal orientation in normal children. Practical implications.

OBJECTIVE: To determine normative anatomical data on the pediatric upper airway. DESIGN: Anatomical study of the trachea and surrounding structures in vivo using magnetic resonance imaging. Three-dimensional positions of the hyoid bone, sternum, skin, and trachea were determined. Tracheal angulation was measured from the vertical plane. SUBJECTS: One hundred children (53 males and 47 females) undergoing magnetic resonance imaging of the brain or chest. INTERVENTION: None. RESULTS: Ages ranged from 2 days to 20.4 years, with a median of 6.05 years. Gender differences were not significant. The mean distance from the hyoid bone to the sternal notch was 63.0 mm (SD, 22.04 mm). The skin to anterior tracheal wall distance averaged 9.34 mm (SD, 3.29 mm), and tracheal diameter averaged 8.48 mm (SD, 2.88 mm). The trachea was not found to be a linear structure. An anterior angulation change of 9.9 degrees (SD, 7.89 degrees) was detected. The point of inflection was found to lie below the sternal notch in children younger than 2 years and above the notch in older children. CONCLUSIONS: Normal anatomical data of the pediatric airway have been acquired using magnetic resonance imaging. Because the trachea exhibits a previously unknown anterior angulation, current design of tracheotomy tubes may not be ideal. Rigid tracheotomy tubes that do not conform to the anatomical structure of the trachea may be responsible for suprastomal collapse or innominate artery erosion.

Selected risk factors in pediatric adenotonsillectomy.

OBJECTIVE: To evaluate the ability of a set of cost-effective criteria to identify before surgery the pediatric patients in whom perioperative respiratory compromise is most likely to develop after adenotonsillectomy. SETTING: A children's hospital medical center. DESIGN: Prospective study using preoperative parental questionnaires and perioperative respiratory status documentation. PATIENTS: All patients scheduled at the outpatient clinic were eligible. MAIN OUTCOME MEASURE: The development of respiratory compromise as defined by at least 1 of the following occurring more than 2 hours after surgery: an oxygen desaturation level of less than 90%, an obstructive breathing pattern, or respiratory distress requiring intervention. RESULTS: The risk of respiratory compromise was significantly increased in patients who were younger than 3 years (P < .001) and in those who had neuromuscular disorders (P < .05), chromosomal abnormalities (P < .005), difficulty in breathing during sleep (P < .005), restless sleep (P < .01), loud snoring with apnea (P < .05), or an upper respiratory tract infection within 4 weeks of surgery (P = .005). Respiratory compromise did not develop in any patients who did not snore (P < .05). CONCLUSIONS: A complete history that includes symptoms suggestive of sleep apnea will assist in the preoperative identification of pediatric patients most at risk for perioperative respiratory compromise after undergoing adenotonsillectomy. Such patients might benefit from overnight observation in a hospital setting. However, when snoring is absent, outpatient surgery is appropriate, as the risk of respiratory compromise is minimal.

Renal biopsy diagnosis of acute lymphocytic leukemia.

Hyperuricemia, due to inborn errors of metabolism, dehydration, or tumor lysis, may cause renal insufficiency. Hyperuricemia from tumor lysis syndrome in malignancy is usually associated with electrolyte disturbances such as hyperkalemia, hyperphosphatemia or hyper or hypocalcemia. Tumor infiltration into the kidneys can occur, yet this accounts for renal insufficiency in only 1% of patients. This infiltration of tumor cells into the kidneys is usually associated with evidence of malignancy elsewhere as identified by physical exam, radiographic studies, and examination of the peripheral smear or bone marrow. We report an unusual presentation of a child with acute lymphocytic leukemia presenting with acute renal failure, nephromegaly and hyperuricemia without electrolyte disturbances or systemic evidence of tumor elsewhere. We stress the importance of kidney biopsy in order to identify the etiology of the renal failure and hyperuricemia.

Innovations in pediatric laryngotracheal reconstruction.

Techniques for the reconstruction of pediatric subglottic and tracheal stenoses continue to undergo modification and refinement. The modified Réthi procedure of anterior laryngotracheal division and posterior cricoidotomy may be supplemented by unilateral or bilateral division of the cricoid cartilage for increased expansion of the subglottic lumen. The resulting four-quadrant division of the cricoid cartilage can be used in conjunction with the placement of cartilaginous grafts and stenting techniques common to laryngotracheal reconstruction (LTR). From October 1, 1986, to January 10, 1990, 31 four-quadrant division procedures were performed in 29 patients. Overall, decannulation has been achieved in 22 of 29 patients (76%). Another technique in LTR is the use of endotracheal tube stenting, resulting in single-stage LTR. The endotracheal tube supports the surgically expanded lumen for a period of intubation, followed by extubation. On extubation, the patient is effectively decannulated because the tracheostomy site is closed during the reconstructive procedure. From January 1, 1985, to July 31, 1990, 36 single-stage LTRs were performed in 35 patients. Overall successful results have been achieved in 30 of 35 (86%) of single-stage LTR patients.