Peter J. Fagenholz

In Vivo Modulation of FGF Biological Activity Alters Cranial Suture Fate

Gain-of-function mutations in fibroblast growth factor receptors have been identified in numerous syndromes associated with premature cranial suture fusion. Murine models in which the posterior frontal suture undergoes programmed fusion after birth while all other sutures remain patent provide an ideal model to study the biomolecular mechanisms that govern cranial suture fusion. Using adenoviral vectors and targeted in utero injections in rats, we demonstrate that physiological posterior frontal suture fusion is inhibited using a dominant-negative fibroblast growth factor receptor-1 construct, whereas the normally patent coronal suture fuses when infected with a construct that increases basic fibroblast growth factor biological activity. Our data may facilitate the development of novel, less invasive treatment options for children with craniosynostosis.

Regional Differentiation of Cranial Suture-Associated Dura Mater In Vivo and In Vitro: Implications for Suture Fusion and Patency

Despite its prevalence, the etiopathogenesis of craniosynostosis is poorly understood. To better understand the biomolecular events that occur when normal craniofacial growth development goes awry, we must first investigate the mechanisms of normal suture fusion. Murine models in which the posterior frontal (PF) suture undergoes programmed sutural fusion shortly after birth provide an ideal model to study these mechanisms. In previous studies, our group and others have shown that sutural fate (i.e., fusion vs. patency) is regulated by the dura mater (DM) directly underlying a cranial suture. These studies have led to the hypothesis that calvarial DM is regionally differentiated and that this differentiation guides the development of the overlying suture. To test this hypothesis, we evaluated the messenger RNA (mRNA) expression of osteogenic cytokines (transforming growth factor β1 [TGF‐β1] and TGF‐β3) and bone‐associated extracellular matrix (ECM) molecules (collagen I, collagen III, osteocalcin, and alkaline phosphatase) in freshly isolated, rat dural tissues associated with the PF (programmed to fuse) or sagittal (SAG; remains patent) sutures before histological evidence of sutural fusion (postnatal day 6 [N6]). In addition, osteocalcin protein expression and cellular proliferation were localized using immunohistochemical staining and 5‐bromo‐2′deoxyuridine (BrdU) incorporation, respectively. We showed that the expression of osteogenic cytokines and bone‐associated ECM molecules is potently up‐regulated in the DM associated with the PF suture. In addition, we showed that cellular proliferation in the DM associated with the fusing PF suture is significantly less than that found in the patent SAG suture just before the initiation of sutural fusion N6. Interestingly, no differences in cellular proliferation rates were noted in younger animals (embryonic day 18 [E18] and N2). To further analyze regional differentiation of cranial suture‐associated dural cells, we established dural cell cultures from fusing and patent rat cranial sutures in N6 rats and evaluated the expression of osteogenic cytokines (TGF‐β1 and fibroblast growth factor 2 [FGF‐2]) and collagen I. In addition, we analyzed cellular production of proliferating cell nuclear antigen (PCNA). These studies confirmed our in vivo findings and showed that dural cell cultures derived from the fusing PF suture expressed significantly greater amounts of TGF‐β1, FGF‐2, and collagen I. In addition, similar to our in vivo findings, we showed that PF suture‐derived dural cells produced significantly less PCNA than SAG suture‐derived dural cells. Finally, coculture of dural cells with fetal rat calvarial osteoblastic cells (FRCs) revealed a statistically significant increase in proliferation (p < 0.001) in FRCs cocultured with SAG suture‐derived dural cells as compared with FRCs cocultured alone or with PF suture‐derived dural cells. Taken together, these data strongly support the hypothesis that the calvarial DM is regionally differentiated resulting in the up‐regulation of osteogenic cytokines and bone ECM molecules in the dural tissues underlying fusing but not patent cranial sutures. Alterations in cytokine expression may govern osteoblastic differentiation and ECM molecule deposition, thus regulating sutural fate. Elucidation of the biomolecular events that occur before normal cranial suture fusion in the rat may increase our understanding of the events that lead to premature cranial suture fusion.

Immature versus Mature Dura Mater: Ii. Differential Expression of Genes Important to Calvarial Reossification

The ability of immature animals and newborns to orchestrate successful calvarial reossification is well described. This capacity is markedly attenuated in mature animals and in humans greater than 2 years of age. Previous studies have implicated the dura mater as critical to successful calvarial reossification. The authors have previously reported that immature, but not mature, dural tissues are capable of elaborating a high expression of osteogenic growth factors and extracellular matrix molecules. These findings led to the hypothesis that a differential expression of osteogenic growth factors and extracellular matrix molecules by immature and mature dural tissues may be responsible for the clinically observed phenotypes (i.e., immature animals reossify calvarial defects; mature animals do not). This study continues to explore the hypothesis through an analysis of transforming growth factor (TGF)-&bgr;3, collagen type III, and alkaline phosphatase mRNA expression. Northern blot analysis of total RNA isolated from freshly harvested immature (n = 60) and mature (n = 10) dural tissues demonstrated a greater than three-fold, 18-fold, and nine-fold increase in TGF-&bgr;3, collagen type III, and alkaline phosphatase mRNA expression, respectively, in immature dural tissues as compared with mature dural tissues. Additionally, dural cell cultures derived from immature (n = 60) and mature dura mater (n = 10) were stained for alkaline phosphatase activity to identify the presence of osteoblast-like cells. Alkaline phosphatase staining of immature dural cells revealed a significant increase in the number of alkaline phosphatase-positive cells as compared with mature dural tissues (p < 0.001). In addition to providing osteogenic humoral factors (i.e., growth factors and extracellular matrix molecules), this finding suggests that immature, but not mature, dura mater may provide cellular elements (i.e., osteoblasts) that augment successful calvarial reossification. These studies support the hypothesis that elaboration of osteogenic growth factors (i.e., TGF-&bgr;3) and extracellular matrix molecules (i.e., collagen type III and alkaline phosphatase) by immature, but not mature, dural tissues may be critical for successful calvarial reossification. In addition, these studies suggest for the first time that immature dural tissues may provide cellular elements (i.e., osteoblasts) to augment this process.

National study of Emergency Department visits for burn injuries, 1993 to 2004.

No studies have examined U.S. burn epidemiology from the perspective of the Emergency Department. We sought to describe patient characteristics, injury types, and Emergency Department practice patterns. Data were collected from the National Hospital Ambulatory Medical Care Survey between 1993 and 2004. Emergency Department visit rates for burn injury decreased from 1993 to 2004 with a peak of 2.8 (95% confidence interval [CI] 2.1–3.4) per 1000 U.S. population in 1995 and a nadir of 1.6 (95% CI 1.2–2.0) per 1000 in 2004. The Emergency Department visit rate for burn injuries was greater for men than women (2.7 [95% CI 2.4–3.0] vs 1.8 [95% CI 1.6–2.0] per 1000) and for black than white subjects (3.4 [95% CI 2.8-3.9] vs 2.1 [95%CI 1.9–2.3] per 1000), though all these groups showed decreases. Emergency Department visit rates for burns were greatest in the first and third decades (3.3 [95% CI 2.8–3.7] and 3.5 [95% CI 3.0–4.0] per 1000, respectively) and decreased thereafter. The upper extremity was the most commonly burned part of the body (37% of total) and most burns of specified depth were partial thickness (48% of total). Less than half of patients received analgesics (47%) or topical antibiotics (38%). Emergency Department visits for burns are declining, but rates remain high in men, black individuals, and children. Burn-prevention efforts should target these groups. Upper-extremity and partial-thickness injuries are common, and less than half of patients receive analgesics or topical antibiotics. Collaboration between burn specialists and Emergency Department personnel should focus on the care of these types of injuries.

Optic nerve sheath diameter correlates with the presence and severity of acute mountain sickness: evidence for increased intracranial pressure

Increased intracranial pressure is suspected in the pathogenesis of acute mountain sickness (AMS), but no studies have correlated it with the presence or severity of AMS. We sought to determine whether increased optic nerve sheath diameter, a surrogate measure of intracranial pressure, is associated with the presence and severity of AMS. We performed a cross-sectional study of travelers ascending through Pheriche, Nepal (4,240 m), from March 3 to May 14, 2006. AMS was assessed using the Lake Louise score. Optic nerve sheath diameter was measured by ultrasound. Ultrasound exams were performed and read by separate blinded observers. Two-hundred eighty seven subjects were enrolled. Ten of these underwent repeat examination. Mean optic nerve sheath diameter was 5.34 mm [95% confidence interval (CI) 5.18-5.51 mm] in the 69 subjects with AMS vs. 4.46 mm (95% CI 4.39-4.54 mm) in the 218 other subjects (P < 0.0001). There was also a positive association between optic nerve sheath diameter and total Lake Louise score (P for trend < 0.0001). In a multivariate logistic regression model of factors associated with AMS, optic nerve sheath diameter was strongly associated with AMS (odds ratio 6.3; 95% CI, 3.7-10.8; P < 0.001). In 10 subjects with repeat examinations, change in Lake Louise score had a strong positive correlation with change in optic nerve sheath diameter (R(2) = 0.84, P < 0.001). Optic nerve sheath diameter, a proxy for intracranial pressure, is associated with the presence and severity of AMS.

Osteoblast Gene Expression is Differentially Regulated by TGF-β Isoforms

The transforming growth factor beta (TGF-&bgr;) superfamily encompasses a number of important growth factors including several TGF-&bgr; isoforms, the bone morphogenetic proteins, activins, inhibins, and growth and differentiation factors. TGF-&bgr;1, -&bgr;2, and -&bgr;3 are three closely related isoforms that are widely expressed during skeletal morphogenesis and bone repair. Numerous studies suggest that each isoform has unique in vivo functions; however, the effects of these TGF-&bgr; isoforms on osteoblast gene expression and maturation have never been directly compared. In the current study, we treated undifferentiated neonatal rat calvaria osteoblast-enriched cell cultures with 2.5 ng/ml of each TGF-&bgr; isoform and analyzed gene expression at 0, 3, 6, and 24 hours. We demonstrated unique isoform-specific regulation of endogenous TGF-&bgr;1 and type I collagen mRNA transcription. To assess the effects of extended TGF-&bgr; treatment on osteoblast maturation, we differentiated osteoblast cultures in the presence of 2.5 ng/ml of each TGF-&bgr; isoform. Analysis of collagen I, alkaline phosphatase, and osteocalcin demonstrated that each TGF-&bgr; isoform uniquely suppressed the transcription of these osteoblast differentiation markers. Interestingly, TGF-&bgr; isoform treatment increased osteopontin expression in primary osteoblasts after 4 and 10 days of differentiation. To our knowledge, these data provide the first direct comparison of the effects of the TGF-&bgr; isoforms on osteoblast gene expression in vitro. Furthermore, these data suggest that TGF-&bgr; isoforms may exert their unique in vivo effects by differentially regulating osteoblast cytokine secretion, extracellular matrix production, and the rate of cellular maturation.

Adequate Nutrition May Get You Home Effect of Caloric/Protein Deficits on the Discharge Destination of Critically Ill Surgical Patients

BACKGROUND Macronutrient deficit in the surgical intensive care unit (ICU) is associated with worse in-hospital outcomes. We hypothesized that increased caloric and protein deficit is also associated with a lower likelihood of discharge to home vs transfer to a rehabilitation or skilled nursing facility. MATERIALS AND METHODS Adult surgical ICU patients receiving >72 hours of enteral nutrition (EN) between March 2012 and May 2014 were included. Patients with absolute contraindications to EN, <72-hour ICU stay, moribund state, EN prior to surgical ICU admission, or previous ICU admission within the same hospital stay were excluded. Subjects were dichotomized by cumulative caloric (<6000 vs ≥ 6000 kcal) and protein deficit (<300 vs ≥ 300 g). Baseline characteristics and outcomes were compared using Wilcoxon rank and χ(2) tests. To test the association of macronutrient deficit with discharge destination (home vs other), we performed a logistic regression analysis, controlling for plausible confounders. RESULTS In total, 213 individuals were included. Nineteen percent in the low-caloric deficit group were discharged home compared with 6% in the high-caloric deficit group (P = .02). Age, body mass index (BMI), Acute Physiology and Chronic Health Evaluation II (APACHE II), and initiation of EN were not significantly different between groups. On logistic regression, adjusting for BMI and APACHE II score, the high-caloric and protein-deficit groups were less likely to be discharged home (odds ratio [OR], 0.28; 95% confidence interval [CI], 0.08-0.96; P = .04 and OR, 0.29; 95% CI, 0.0-0.89, P = .03, respectively). CONCLUSIONS In surgical ICU patients, inadequate macronutrient delivery is associated with lower rates of discharge to home. Improved nutrition delivery may lead to better clinical outcomes after critical illness.

Systemic local anaesthetic toxicity from continuous thoracic paravertebral block

Continuous paravertebral block is commonly used for post-thoracotomy analgesia and compares favourably with other systemic and regional methods with regard to safety and efficacy. No major complications of continuous paravertebral block for post-thoracotomy analgesia have been reported previously. We report here a case of systemic local anaesthetic toxicity from continuous paravertebral block administration after thoracotomy and lobectomy leading to seizure, aspiration, and ultimately, death. Potential contributing factors in this case included small patient size, concomitant antifungal therapy, extensive surgical disruption of the pleurae, and inappropriate paravertebral bolus administration. Postoperative delirium was a diagnostic confounder. We discuss the potential causes and means of avoiding similar complications in the future.

Fulminant Clostridium difficile colitis: prospective development of a risk scoring system.

BACKGROUND Of the patients with a Clostridium difficile infection, 2% to 8% will progress to fulminant C. difficile colitis (fCDC), which carries high morbidity and mortality. No system exists to rapidly identify patients at risk for developing fCDC and possibly in need of surgical intervention. Our aim was to design a simple and accurate risk scoring system (RSS) for daily clinical practice. METHODS We prospectively enrolled all patients diagnosed with a C. difficile infection and compared patients with and without fCDC. An expert panel, combined with data derived from previous studies, identified four risk factors, and a multivariable logistic regression model was performed to determine their effect in predicting fCDC. The RSS was created based on the predictive power of each factor, and calibration, discrimination, and test characteristics were subsequently determined. In addition, the RSS was compared with a previously proposed severity scoring system. RESULTS A total of 746 patients diagnosed with C. difficile infection were enrolled between November 2010 and October 2012. Based on the log (odds ratio) of each risk factor, age greater than 70 years was assigned 2 points, white blood cell count equal to or greater than 20,000/&mgr;L or equal to or less than 2,000/&mgr;L was assigned 1 point, cardiorespiratory failure was assigned 7 points, and diffuse abdominal tenderness on physical examination was assigned 6 points. With the use of this system, the discriminatory value of the RSS (c statistic) was 0.98 (95% confidence interval, 0.96–1).The Hosmer-Lemeshow goodness-of-fit test showed a p value of 0.78, and the Brier score was 0.019. A value of 6 points was determined to be the threshold for reliably dividing low-risk ( <6) from high-risk (≥6) patients. CONCLUSION The RSS is a valid and reliable tool to identify at the bedside patients who are at risk for developing fCDC. External validation is needed before widespread implementation. LEVEL OF EVIDENCE Prognostic study, level II.

National study of United States emergency department visits for acute pancreatitis, 1993-2003.

BackgroundThe epidemiology of acute pancreatitis in the United States is largely unknown, particularly episodes that lead to an emergency department (ED) visit. We sought to address this gap and describe ED practice patterns.MethodsData were collected from the National Hospital Ambulatory Medical Care Survey between 1993 and 2003. We examined demographic factors and ED management including medication administration, diagnostic imaging, and disposition.ResultsED visits for acute pancreatitis increased over the study period from the 1994 low of 128,000 visits to a 2003 peak of 318,000 visits (p = 0.01). The corresponding ED visit rate per 10,000 U.S. population also increased from 4.9 visits (95%CI, 3.1–6.7) to 10.9 (95%CI, 7.6–14.3) (p = 0.01). The average age for patients making ED visits for acute pancreatitis during the study period was 49.7 years, 54% were male, and 27% were black. The ED visit rate was higher among blacks (14.7; 95%CI, 11.9–17.5) than whites (5.8; 95%CI, 5.0–6.6). At 42% of ED visits, patients did not receive analgesics. At 10% of ED visits patients underwent CT or MRI imaging, and at 13% of visits they underwent ultrasound testing. Two-thirds of ED visits resulted in hospitalization. Risk factors for hospitalization were older age (multivariate odds ratio for each increasing decade 1.5; 95%CI, 1.3–1.8) and white race (multivariate odds ratio 2.3; 95%CI, 1.2–4.6).ConclusionED visits for acute pancreatitis are rising in the U.S., and ED visit rates are higher among blacks than whites. At many visits analgesics are not administered, and diagnostic imaging is rare. There was greater likelihood of admission among whites than blacks. The observed race disparities in ED visit and admission rates merit further study.