Exploiting PI3K/mTOR signaling to accelerate epithelial wound healing

The molecular circuitries controlling the process of skin wound healing have gained new significant insights in recent years. This knowledge is built on landmark studies on skin embryogenesis, maturation, and differentiation. Furthermore, the identification, characterization, and elucidation of the biological roles of adult skin epithelial stem cells and their influence in tissue homeostasis have provided the foundation for the overall understanding of the process of skin wound healing and tissue repair. Among numerous signaling pathways associated with epithelial functions, the PI3K/Akt/mTOR signaling route has gained substantial attention with the generation of animal models capable of dissecting individual components of the pathway, thereby providing a novel insight into the molecular framework underlying skin homeostasis and tissue regeneration. In this review, we focus on recent findings regarding the mechanisms involved in wound healing associated with the upregulation of the activity of the PI3K/Akt/mTOR circuitry. This review highlights critical findings on the molecular mechanisms controlling the activation of mTOR, a downstream component of the PI3K–PTEN pathway, which is directly involved in epithelial migration and proliferation. We discuss how this emerging information can be exploited for the development of novel pharmacological intervention strategies to accelerate the healing of critical size wounds.     

Construction of a Tissue‐Engineered Annulus Fibrosus

The intervertebral disc is composed of load‐bearing fibrocartilage that may be subjected to compressive forces up to 10 times the body weight. The multilaminated outer layer, the annulus fibrosus (AF), is vulnerable to damage and its regenerative potential is limited, sometimes leading to nuclear herniation. Scaffold‐based tissue engineering of AF using stem cell technology has enabled the development of bi‐laminate constructs after 10 weeks of culture. It is difficult to know if these constructs are limited by the differentiation state of the stem cells or the culture system. In this study, we have characterized an expandable scaffold‐free neoconstruct using autologous AF cells. The construct was prepared from pellet cultures derived from monolayer cultures of AF cells from mature pigs that became embedded in their own extracellular matrix. The pellet cultures were incubated for 24 h in a standardized conical tube and then carefully transferred intact to a culture flask and incubated for 21 days to allow continued matrix synthesis. Cell viability was maintained above 90% throughout the culture period. The engineered scaffold‐free construct was compared with the native AF tissue by characterization of gene expression of representative markers, histological architecture, and biochemical composition. The morphological and biochemical characteristics of the cultured disc construct are very similar to that of native AF. The cell number per gram of construct was equal to that of native AF. Expression of aggrecan was elevated in the engineered construct compared with RNA extracted from the AF. The glycosaminoglycan content in the engineered construct showed no significant difference to that from native construct. These data indicate that scaffold‐free tissue constructs prepared from AF cells using a pellet‐culture format may be useful for in vitro expansion for transplantation into damaged discs.     

Local Immune Response Predicts Survival in Patients with Thick (T4) Melanomas

Background

Tumor infiltrating lymphocytes (TIL) and histological regression in primary melanoma are generally considered indicators of the local immune response but their roles as prognostic factors have been variably reported. We examined the prognostic role of these variables in patients with high risk (T4) primary melanomas in a large series of patients with long-term follow-up.

Methods

From a prospectively maintained cohort of patients diagnosed between 1971 and 2004, 161 patients were retrospectively identified with primary thick melanomas (>4 mm), no clinical evidence of regional nodal disease (RND) at diagnosis and complete histopathologic data. Univariate and multivariate Cox regression models were performed to identify clinical and histopathologic predictors of disease-specific survival (DSS) and to identify subgroups with differential survival.

Results

Factors significantly associated with decreased DSS by univariate analysis included male gender, age ≥ 60 years, axial anatomic location, presence of ulceration, RND, absence of TIL, and presence of regression. In the final multivariate model, TIL and regression, as interacting variables, and RND status remained significantly associated with DSS. In the presence of TIL, concomitant regression was associated with significantly worse survival (p ≤ 0.0001). In the absence of TIL, there was no effect of regression on survival (p = 0.324).

Conclusions

Primary TIL and regression status and RND status are independently associated with melanoma-specific survival in patients with T4 melanomas; presence of TIL in the primary melanoma with concomitant radial growth phase regression is associated with a poor prognosis and may reflect an ineffective local regional immune response.     

Intraoperative neurophysiologic monitoring and neurologic outcomes in patients with epidural spine tumors

Purpose

Multimodal intraoperative neurophysiologic monitoring (IOM) provides assessment of spinal cord pathways during neurosurgery. Despite widespread use, few data exist regarding sensitivity and specificity of IOM in predicting neurologic outcome during decompression and instrumentation for epidural spine tumors.

Methods

Retrospective analysis evaluated consecutive spine procedures involving IOM modalities (somatosensory evoked potentials [SSEP], motor evoked potentials [MEP], and electromyography [(EMG]) from 2007 to 2009. Demographic and surgical information, intraoperative neurophysiologic data, and pre- and postoperative neurologic status were collected. All cases involved neoplastic epidural spinal cord compression by a primary or metastatic tumor and included posterolateral decompression and instrumented fusion.

Results

Two-hundred and eight consecutive patients had spine surgery during this time period and one hundred and fifty-two met inclusion criteria. All patients had SSEP monitoring, with 4 having transient changes and 7 persistent changes. One hundred and twenty-two patients had combined SSEP and MEP monitoring, with 3 having transient changes and 4 persistent changes in MEP signals. Two patients had neurophysiologic changes associated with hypotension and correction led to normalization. One developed new neurologic deficits after surgery. Two from the total cohort had new postoperative neurologic deficits. One had a transient decrease in MEP amplitude while the other had no intraoperative changes.

Discussion

These cases are often long with significant blood loss, and stability of multiple IOM modalities provides reassurance that spinal cord function remains intact. Signal changes should result in scrutiny of blood pressure, surgical technique and anesthesia. Preserved IOM signals are suggestive of preserved neurologic outcome.     

Prevalence of CYP1B1 mutations in Australian patients with primary congenital glaucoma

Analysis of CYP1B1 in primary congenital glaucoma (PCG) patients from various ethnic populations indicates that allelic heterogeneity is high, and some mutations are population specific. No study has previously reported the rate or spectrum of CYP1B1 mutations in Australian PCG patients. The aim of this study is to determine the frequency of CYP1B1 mutations in our predominately Caucasian, Australian cohort of PCG cases. Thirty-seven probands were recruited from South-Eastern Australia, along with 100 normal control subjects. Genomic DNA was extracted and the coding regions of CYP1B1 analysed by direct sequencing. Sequence analysis identified 10 different CYP1B1 disease-causing variants in eight probands (21.6%). Five subjects were compound heterozygotes, two subjects heterozygous and one homozygous for CYP1B1 mutations. Three missense mutations are novel (D192Y, G329D, and P400S). None of the novel mutations identified were found in normal controls. One normal control subject was heterozygous for the previously reported CYP1B1 R368H mutation. Six previously described probable polymorphisms were also identified. Mutations in CYP1B1 account for approximately one in five PCG cases from Australia. Our data also supported the high degree of allelic heterogeneity seen in similar studies from other ethnic populations, thereby underscoring the fact that other PCG-related genes remain to be identified.     

An ELISA for plasma retinol-binding protein using monoclonal and polyclonal antibodies: plasma variation in normal and insulin resistant subjects

OBJECTIVES: Plasma retinol-binding protein (RBP) has been linked to insulin resistance and cardiovascular risk, yet little is know of its natural variation in plasma. We examined this in normal subjects and compared plasma levels and variability in lean subjects and subjects with the metabolic syndrome. METHODS: We established an "in house" ELISA for plasma RBP and measured levels in 20 normal subjects over daylight hours and 2 subject groups, either lean or classified with the metabolic syndrome. RESULTS: Plasma RBP in normal subjects did not vary over the day with no differences between males and females. There was also no difference in plasma RBP levels and between the age- and sex-matched lean subjects compared to the metabolic syndrome group. CONCLUSION: The lack of variation in plasma RBP in normal subjects and the lack of difference between plasma RBP in normal and metabolic syndrome subjects suggest the link between plasma RBP and insulin resistance is tenuous. Investigating a large cohort over the diabetic non-diabetic spectrum may clarify this issue.