Embryology of the upper limb: the molecular orchestration of morphogenesis.

Accumulating data on the molecular interactions that occur during limb development have greatly enhanced our understanding of the process of limb morphogenesis. In this chapter, the key morphologic events are described, the broad categories of molecules involved are defined, the known molecular cascades and specific pathways that orchestrate limb development are reviewed. In addition, cascades disrupted by known genetic mutations associated with limb malformations are identified.

Enhanced therapeutic effect of HSV-tk+GCV gene therapy and ionizing radiation for prostate cancer.

Standard therapies for prostate cancer including radiation, prostatectomy, and hormone ablation have significant toxicities and recurrence risk. HSV-tk gene therapy may be effective in combination with radiation therapy due to complementary mechanisms and distinct toxicity profiles. Mouse prostate tumors transplanted subcutaneously were treated by either gene therapy involving intratumoral injection of AdV-tk followed by systemic ganciclovir or local radiation therapy or the combination of gene and radiation therapy. Both single-therapy modalities showed a 38% decrease in tumor growth compared to controls. The combined treatment resulted in a decrease of 61%. In addition the combined-therapy group had a mean survival of 22 days versus 16.6 days for single therapy and 13.8 days for nontreated controls. To analyze systemic anti-tumor activity, lung metastases were generated by tail vein injection of RM-1 prostate cancer cells on the same day that they were injected subcutaneously. The primary tumors were treated as before with AdV-tk followed by ganciclovir, radiation, or the combination. The number of lung nodules was reduced by 37% following treatment with AdV-tk, whereas radiotherapy alone had no effect on metastatic growth. The combination led to an additional 50% reduction in lung colonization. Primary tumors that received the combination therapy had a marked increase in CD4 T cell infiltrate. This is the first report showing a dramatic systemic effect following the local combination treatment of radiation and AdV-tk. A clinical study using this strategy has been initiated and patient accrual is ongoing.

Regulation of glomerular basement membrane collagen expression by LMX1B contributes to renal disease in nail patella syndrome.

Basement membrane (BM) morphogenesis is critical for normal kidney function. Heterotrimeric type IV collagen, composed of different combinations of six alpha-chains (1-6), is a major matrix component of all BMs (ref. 2). Unlike in other BMs, glomerular BM (GBM) contains primarily the alpha 3(IV) and alpha 4(IV) chains, together with the alpha 5(IV) chain. A poorly understood, coordinated temporal and spatial switch in gene expression from ubiquitously expressed alpha 1(IV) and alpha 2(IV) collagen to the alpha 3(IV), alpha 4(IV) and alpha 5(IV) chains occurs during normal embryogenesis of GBM (ref. 4). Structural abnormalities of type IV collagen have been associated with diverse biological processes including defects in molecular filtration in Alport syndrome, cell differentiation in hereditary leiomyomatosis, and autoimmunity in Goodpasture syndrome; however, the transcriptional and developmental regulation of type IV collagen expression is unknown. Nail patella syndrome (NPS) is caused by mutations in LMX1B, encoding a LIM homeodomain transcription factor. Some patients have nephrosis-associated renal disease characterized by typical ultrastructural abnormalities of GBM (refs. 8,9). In Lmx1b(-/-) mice, expression of both alpha(3)IV and alpha(4)IV collagen is strongly diminished in GBM, whereas that of alpha1, alpha2 and alpha5(IV) collagen is unchanged. Moreover, LMX1B binds specifically to a putative enhancer sequence in intron 1 of both mouse and human COL4A4 and upregulates reporter constructs containing this enhancer-like sequence. These data indicate that LMX1B directly regulates the coordinated expression of alpha 3(IV) and alpha 4(IV) collagen required for normal GBM morphogenesis and that its dysregulation in GBM contributes to the renal pathology and nephrosis in NPS.

LMX1B transactivation and expression in nail-patella syndrome.

Lmx1b, a member of the LIM homeodomain protein family, is essential for the specification of dorsal limb fates at the zeugopodal and autopodal level in vertebrates. We and others have shown that a skeletal dysplasia, nail-patella syndrome (NPS), results from mutations in LMX1B. While it is a unique mesenchymal determinant of dorsal limb patterning during vertebrate development, the mechanism by which LMX1B mutations generate the NPS phenotype has not been addressed at a transcriptional level or correlated with its spatial pattern of gene expression. In this study, in situ hybridizations of Lmx1b on murine limb sections reveal strong expression in dorsal mesenchymal tissues (precursors of muscle, tendons, joints and patella) and, interestingly, also in anterior structures of the limb, explaining the anterior to posterior gradient of joint and nail dysplasia observed in NPS patients. Transfection studies showed that both the LIM domain-interacting protein, LDB1, and the helix-loop-helix protein, E47/shPan1, can regulate LMX1B action. While co--transfections of E47/shPan1 with LMX1B result in a synergistic effect on reporter activity, LDB1 down-regulated LMX1B-mediated transactivation irrespective of E47/shPan1. Mutant LMX1B proteins containing human mutations affecting each of the helices or the N-terminal arm of the homeodomain abolished transactivation, while LIM B and truncation mutations retained residual activity. These mutations fail to act in a dominant-negative manner on wild-type LMX1B in mixing studies, thereby supporting haploinsufficiency as the mechanism underlying NPS pathogenesis.

Vinorelbine tartrate-induced pulmonary edema confirmed on rechallenge.

A 67-year-old woman with metastatic breast cancer experienced sudden and profound pulmonary edema within 45 minutes after completion of intravenous administration of vinorelbine tartrate on two occasions. Both times the drug was discontinued and the patient was treated aggressively with oxygen, intravenous furosemide, and a vasodilator. The patient suffered no lasting medical complications due to the reaction. Until clear documentation and the mechanism for occurrence of this reaction are known, patients receiving vinorelbine should be monitored closely, particularly in the first few hours after intravenous administration.

Endoscopic intrauterine surgery in primates: overcoming technical obstacles.

Current protocols for fetal surgery require cesarean section and partial fetal extraction, both of which impart significant risks to the mother and fetus. Endoscopic fetal surgery is less invasive and will likely reduce some of these risks, but the technical difficulties and feasibility in a primate model have yet to be explored fully. Four pregnant baboons (95 days gestation) were anesthetized, their uteruses exposed via an abdominal incision, and blunt-tipped flanged endoscopic ports inserted. Amniotic fluid was removed, and warmed saline was infused to dilate the uterus. To evaluate instrumentation and wound closure, the tip of the snout was externalized and bilateral cleft lip-like defects made. The lips were then endoscopically repaired by suture (Endostitch, U.S. Surgical) or unique nonpenetrating clips (VCS, U.S. Surgical). The saline was then removed, amniotic fluid returned, and the ports carefully removed. After 4 weeks, the fetuses were delivered and evaluated. Eight cleft lip-like defects were successfully repaired in all four cases. Operative time averaged 83 min. No infections, amniotic leaks, or adhesions developed. Survival was 50% with two fetuses delivering within 48 hours postoperatively: one from preterm labor, the other with fetal demise from retroperitoneal hemorrhage after operative blunt abdominal trauma. We demonstrate the feasibility of endoscopic fetal surgery in primates. The use of blunt-tipped flanged ports provides a fluid tight seal and allows appropriate closure of the fetal membranes, but requires laparotomy and uterine exposure. Distension of the uterus with warmed saline affords a larger operating field, enhancing visualization and instrumentation of the fetus. Grasping the fetus through the exposed uterus gives excellent control for repair. However, such control is also needed in a percutaneous approach. Further instrumentation development is needed to accomplish similar control for the percutaneous approach.

Judicious evaluation of adverse drug reactions: inaccurate assessment of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor-induced muscle injury.

Adverse reactions in two patients who received HMG CoA reductase inhibitor therapy were reinvestigated because of their rarity. A case of permanent forearm myalgia was thought to be caused by atorvastatin. Closer evaluation and work-up revealed underlying lateral epicondylitis, and atorvastatin was not considered the cause of the disability. In another patient, rhabdomyolysis was suspected to be secondary to simvastatin. However, after an extensive review, the reaction was believed to be compartment syndrome of the anterior tibial area. An adverse drug reaction report requires careful and judicious assessment to assign the correct probability for the event.

Renal tubular dysgenesis in twin-twin transfusion syndrome.

In twin-twin transfusion syndrome (TTTS), the disparity in circulation is reflected in discordant fetal growth, urine output, and amniotic fluid accumulation. The effect of uneven shunting of the growth factor and nutrient-rich vasculature on development and differentiation of the kidney has not been well studied. We analyzed renal tubular growth and differentiation in 25 fetal autopsies with TTTS (13 donors and 12 recipients, including 9 sibling pairs) between 18 and 33 weeks gestation. Immunohistochemical markers for fumarylacetoacetate hydrolase (FAH), Leu-M1, and Lotus tetragonolobus (LTA) were used to identify proximal convoluted tubules, and epithelial membrane antigen (EMA) was used to demonstrate distal convoluted and collecting tubules. FAH appeared to be more specific and reliable than either Leu-M1 or LTA in the identification of proximal tubules. Donors tended to demonstrate a paucity of proximal tubules with crowding of glomeruli characteristic of renal tubular dysgenesis (RTD). The degree of dysgenesis was greater in later gestations and associated with more severe growth restriction. Donors in TTTS are at risk for the development of RTD. Several authors suggest ischemia as the underlying cause of "acquired" RTD. However, in this setting there is no evidence of cell death or necrosis, and we suggest that hypoperfusion leading to decreased glomerular filtration is the underlying etiology, with the severity of RTD related to the degree of shunting.

Cardiac patient air medical transport in the northeast region of the United States.

OBJECTIVES: 1) To develop and analyze a database of cardiac patient demographics and transport variables in the northeast region of the United States, and 2) to develop skills to enable future collaborative research in this region. METHOD: A retrospective review of air transport medical records was performed by flight crew members from five independent air medical transport programs. Parameters describing age, gender, cardiac diagnosis, referral patterns, hemodynamic parameters, medication infusions, arrhythmias, and invasive procedures were included. RESULTS: Data for 1,320 cardiac patients over the age of 30 years were compiled. Seventy percent were male, 30% were female, and the mean age was 60.7 years. The median flight time was 13 minutes. Seventy-three percent had a referring agency diagnosis of acute myocardial infarction. Transport originated from an emergency department 49% of the time and from an intensive care unit 51% of the time. Twenty percent of the patients had a heart rhythm other than normal sinus rhythm during transport. Twenty-eight patients had ventricular arrhythmias in flight (2%) and 28 patients suffered cardiac arrests (2%). Cardiogenic shock was observed in 13% of the patients. Twenty percent of the patients were endotracheally intubated. CONCLUSIONS: A database of patients transported by air for cardiac diagnoses was successfully established. Acute myocardial ischemia was the major indication for air transport. A region of flight programs collaborating on a research project can generate a large pool of subjects for analysis. Limitations of the methodology were identified and future research goals were better defined.

Prenatal ultrasonographic description and postnatal pathological findings in atelosteogenesis type 1.

Atelosteogenesis type 1 (AO1) is a rare lethal chondrodysplasia characterized by incomplete ossification of cartilage anlagen. Histologically, the cartilage contains irregular clusters that occasionally include giant chondrocytes. Pulmonary hypoplasia is a characteristic finding that has been presumed to be the cause of neonatal lethality. We report on a male fetus with AO1 and document the early ultrasonographic/ radiologic progression of this disorder from 15 weeks gestation until delivery at 41 weeks. While the radiological findings we describe are typical of AO1 by the lack of proximal and middle phalangeal ossification, the complete radiological picture showed considerable overlap with boomerang dysplasia. Although pulmonary hypoplasia was present, it was moderate and considered unlikely to be the sole cause of death. Detailed neonatal and postmortem examination showed severe subglottic hypoplasia and tracheomalacia. The tracheal walls were supported by thin and pliable cartilaginous plates that allowed luminal collapse with minimal pressure. The marked luminal narrowing, tracheomalacia, and temporal proximity of extubation to demise support tracheal collapse as a major contributor to the death in AO1. The detailed description of this patient should contribute to earlier diagnosis of this condition; anticipation of the poor prognosis in AO1 is essential for appropriate genetic counseling of the parents and for determining postnatal treatment options.

Erythematous bullous lesions on the dorsa of the hands due to contact exposure to ibutilide fumarate for injection.

While waiting to observe the response to ibutilide fumarate by a patient with atrial fibrillation, a nurse preparing the intravenous solution inadvertently spilled the drug on the hands of a medical resident. The resident immediately wiped his hands dry with disposable paper towels. Several hours later he sensed tingling and itching over the area, and the next day two erythematous bullous lesions were present on the dorsal surfaces of both hands. A single application of topical steroid was applied to the affected areas. The lesions were kept clean and dry, and healed completely in approximately 10 days. This is an unusual allergic reaction due to contact with ibutilide fumarate.

Endoscopic excision and repair of simulated bilateral cleft lips in fetal lambs.

The use of nonpenetrating clips to accomplish wound closure as an alternative to suture in the repair of simulated cleft lips in partially exteriorized fetuses has been described previously. In this study, the fetus is approached endoscopically, and clipped (n = 8) and sutured (n = 4) intrauterine endoscopic repairs in six lambs (90- to 95-day gestation) are compared. Also used was a newly developed harmonic scalpel to create the defects in the fluid environment. Clipped repairs were nearly 10 times faster than sutured repairs (2.7 +/- 0.5 minutes compared with 24 +/- 4 minutes, respectively). Furthermore, suture incited foreign body inflammation, recruited monocytic inflammatory cells, and exhibited notable scarring. The comparison between clipped and sutured repairs extends the previous observations to the realm of endoscopy and reinforces the previous conclusions of this group that the nonpenetrating clip is more rapid and incites less inflammation than suture in fetal wound approximation and repair.

Limb and kidney defects in Lmx1b mutant mice suggest an involvement of LMX1B in human nail patella syndrome.

Dorsal-ventral limb patterning in vertebrates is thought to be controlled by the LIM-homeodomain protein Lmx1b which is expressed in a spatially and temporally restricted manner along the dorsal-ventral limb axis. Here we describe the phenotype resulting from targeted disruption of Lmx1b. Our results demonstrate that Lmx1b is essential for the specification of dorsal limb fates at both the zeugopodal and autopodal level with prominent phenotypes including an absence of nails and patellae. These features are similar to those present in a dominantly inherited human condition called nail patella syndrome (NPS), which also has renal involvement. Mouse Lmx1b maps to a region syntenic to that of the NPS gene, and kidneys of Lmx1b mutant mice exhibit pathological changes similar to that observed in NPS (refs 5,6). Our results demonstrate an essential function for Lmx1b in mouse limb and kidney development and suggest that NPS might result from mutations in the human LMX1B gene.

Delayed elevation of international normalized ratio with concurrent clarithromycin and warfarin therapy.

Clarithromycin is rarely reported to cause disturbances in anticoagulation. Theoretically, clarithromycin administered concurrently with warfarin could result in enhanced anticoagulation, since many properties of clarithromycin are similar to those of erythromycin, which interacts with warfarin. A search of the National Library of Medicine produced no published reports of an interaction between the drugs. Erythromycin competitively inhibits hepatic metabolism of warfarin, specifically the R-warfarin enantiomer, by the cytochrome P450 3A3 and 3A4 pathways, resulting in increased prothrombin time and international normalized ratio (INR). Two men, age 61 and 70 years, who received stable warfarin regimens, experienced supratherapeutic elevations in prothrombin time (98.4 and 26.8 sec) and INR (90.3 and 5.6), respectively. While taking warfarin, both patients experienced dramatically increased anticoagulation effects 5 days after starting clarithromycin for atypical pneumonia. They were similarly managed for overanticoagulation by discontinuing clarithromycin, holding warfarin, and receiving intravenous phytonadione. Neither man suffered from medical complications related to anticoagulation. Until clear documentation of the exact mechanism and temporal relationship of this interaction is known, patients receiving warfarin who require the concurrent clarithromycin should have prothrombin time and INR closely monitored.

Medroxyprogesterone acetate in the treatment of obstructive sleep apnea.

The use of MPA in patients with OSA is limited. Instead, patients with OSA should be encouraged to abstain from alcohol and respiratory depressive agents, and avoid sleeping in the supine position. In general, patients with OSA are most effectively treated with CPAP and/or surgery. Patients need to be encouraged to maintain ideal body weight, since this has been associated with a marked reduction in symptoms. For patients who are not surgical candidates or refuse to use CPAP, drug therapy may be beneficial. Fluoxetine has been shown to be as effective as protriptyline, and is better tolerated. However, further study is needed to determine whether selective serotonin-reuptake inhibitors are beneficial in treating OSA. Therefore, MPA therapy should be reserved for hypercapnic patients who refuse other modalities of treatment. The potential long-term adverse effects of MPA must be addressed before initiating therapy in men with OSA.

Histopathologic evaluation of the canine prostate following electrovaporization.

PURPOSE: Transurethral electrovaporization of the prostate (TVP) for symptomatic benign prostatic hypertrophy (BPH) has proven to be efficacious with minimal patient morbidity. When compared to transurethral resection of the prostate (TURP), TVP demonstrates comparable postoperative flow rates, American Urologic Association (AUA) symptom score indices, and a potential cost savings. However, in the human studies it has not been possible to correlate these clinical parameters with procedure-related histopathologic changes in the prostate immediately postoperative or during wound healing. The following study was done using a canine model in an effort to evaluate these histopathologic changes. METHODS AND MATERIALS: Fifteen hounds (25-35 kg.) underwent antegrade electrovaporization of the prostate, via an open cystotomy, using a Circon ACMI USA series resectoscope and video equipment. The dogs were sacrificed and the prostates harvested at various intervals postoperatively (0-11 weeks). The prostates were evaluated grossly as well as histologically for cavitary defects, depth of necrosis, and cellular response. RESULTS: Prostates examined immediately following the procedure demonstrated superficial necrosis (less than 2 mm.) in the region of vaporized tissue. One week postoperatively, the vaporized regions demonstrated an intense acute inflammation amidst superficial necrosis with focal hemorrhage and dystrophic calcification. Transient glandular cystic changes developed, but were resolving by seven weeks postoperatively. Re-epithelialization was underway by the third postoperative week and epithelial stratification underway by the fifth week. There was no extension of the initial two millimeter zone of necrosis at any time point examined. CONCLUSION: TVP in the canine model vaporizes prostatic tissue at the site of contact. Only a shallow remnant of necrosis remains at the site of vaporization, indicating the highly localized effect of this technique. Healing at the site of vaporization occurs in a rapid and expected manner. These data provide a histopathologic rationale for the minimal morbidity and the efficacious nature of this technique demonstrated in clinical studies.

QT interval prolongation and torsades de pointes due to erythromycin lactobionate.

STUDY OBJECTIVES: To discern the frequency of torsades de pointes and QT prolongation in patients receiving intravenous erythromycin lactobionate; to examine the degree of QT prolongation and QT dispersion due to intravenous erythromycin in a typical clinical setting; and to identify any concurrent factors that might predispose patients to excessive QT prolongation or torsades de pointes while receiving intravenous erythromycin. DESIGN: Retrospective cohort trial. SETTING: A university teaching hospital. PATIENTS: All inpatients who received intravenous erythromycin lactobionate during a 1-year period. MEASUREMENTS AND MAIN RESULTS: The records of 278 consecutive patients were analyzed, of whom 49 had 12-lead electrocardiograms while receiving and not receiving erythromycin. The dosages of erythromycin ranged from 18-83 (42 +/- 18) mg/kg/day. Of the 49 patients, the baseline QTc was 432 +/- 39 msec, compared with 483 +/- 62 msec during erythromycin therapy (p < 0.01). In 30 of 49 patients with heart disease, the increase in QTc due to erythromycin was 15 +/- 11%, compared with 8.6 +/- 10% in the 19 patients without heart disease (p < 0.05). The degree of QTc dispersion was 34 +/- 16 msec at baseline, compared with 80 +/- 35 msec with erythromycin (p < 0.01). Overall, 19 (39%) of 49 patients had a moderate to severe delay in ventricular repolarization (QTc > or = 500 msec). Of the 278 patients prescribed intravenous erythromycin over the year, it caused torsades de pointes in just one (< or = 0.4%). CONCLUSION: Erythromycin lactobionate-induced torsades de pointes is rare, although QT prolongation is common. Some patients may be at risk for suffering torsades de pointes due to this agent, particularly if heart disease or other factors that may further delay ventricular repolarization are present.