Pigmentation Diathesis of Hypertrophic Scar: An Examination of Known Signaling Pathways to Elucidate the Molecular Pathophysiology of Injury-Related Dyschromia.

Hypertrophic scar (HTS) occurs frequently after burn injury. Treatments for some aspects of scar morbidity exist, however, dyspigmentation treatments are lacking due to limited knowledge about why scars display dyschromic phenotypes. Full thickness wounds were created on duroc pigs that healed to form dyschromic HTS. HTS biopsies and primary cell cultures were then used to study pigmentation signaling. Biopsies of areas of both pigment types were taken for analysis. At the end of the experiment, melanocyte-keratinocyte cocultures were established from areas of differential pigmentation. Heterogeneously dyspigmented scars formed with regions of hyperpigmentation and hypopigmentation. Melanocytes were present in both pigment types measured by S100ß quantitative real time-polymerase chain reaction (qRT-PCR) and immunostaining, and visualized by dendritic cell presence in primary cultures. P53 expression was not different between the two pigment types. Hyperpigmented scars had upregulated levels of proopiomelanocortin (POMC), adrenocorticotropic hormone (ACTH), α-melanocyte stimulating hormone (α-MSH), stem cell factor (SCF), and c-KIT and melanocortin 1 receptors (MC1R) compared to hypopigmented regions. Many genes involved in dyspigmentation were differentially regulated by microarray analysis including MITF, TYR, TYRP1, and DCT. MiTF expression was not different upon further exploration, but TYR, TYRP1, and DCT were upregulated in intact biopsies measured by qRT-PCR and confirmed by immunostaining. This is the first work to confirm the presence of melanocytes in hypopigmented scar using qRT-PCR and primary cell culture. An understanding of the initial steps in dyspigmentation signaling, as well as the downstream effects of these signals, will inform treatment options for patients with scars and provide insight to where pharmacotherapy may be directed.

Analysis of factor XIa, factor IXa and tissue factor activity in burn patients.

INTRODUCTION: An elevated procoagulant activity observed in trauma patients is, in part, related to tissue factor (TF) located on blood cells and microparticles. However, analysis of trauma patient plasma indicates that there are other contributor(s) to the procoagulant activity. We hypothesize that factor (F)XIa and FIXa are responsible for an additional procoagulant activity in burn patients. METHODS: Multiple time-point plasma samples from 56 burn patients (total number of samples was 471; up to 20 time-points/patient collected in 3 weeks following admission) were evaluated in a thrombin generation assay using inhibitory antibodies to TF, FIXa and FXIa. RESULTS: Due to the limited volume of some samples, not all were analyzed for all three proteins. At admission, 10 of 53 patients (19%) had active TF, 53 of 55 (96%) had FXIa and 48 of 55 (87%) had FIXa in their plasma. 34 patients of 56 enrolled (61%) showed TF activity at one or more time-points. All patients had FXIa and 96% had FIXa at one or more time-points. Overall, TF was observed in 99 of 455 samples analyzed (22%), FXIa in 424 of 471 (90%) and FIXa in 244 of 471 (52%). The concentration of TF was relatively low and varied between 0 and 2.1pM, whereas that of FXIa was higher, exceeding 100pM in some samples. The majority of samples with FIXa had it at sub-nanomolar concentrations. No TF, FXIa and FIXa activity was detected in plasma from healthy individuals. CONCLUSIONS: For the first time reported, the majority of plasma samples from burn patients have active FXIa and FIXa, with a significant fraction of them having active TF. The concentration of all three proteins varies in a wide range.

Effectiveness of a Glycylcycline Antibiotic for Reducing the Pathogenicity of Superantigen-Producing Methicillin-Resistant Staphylococcus aureus in Burn Wounds.

Objective: Burn-injured patients are highly susceptible to infectious complications, which are often associated with increased morbidity and mortality. Rates of antibiotic resistance have increased, and resistant species such as methicillin-resistant Staphylococcus aureus provide additional challenges in the form of virulence factors. Proteins can disrupt local healing, leading to systemic immune disruption. To optimize outcomes, treatments that reduce pathogenicity must be identified. This study aims to compare a glycylcycline antibiotic-tigecycline-with clindamycin for effectiveness in treating superantigenic methicillin-resistant Staphylococcus aureus in burn wounds. Methods: Sprague-Dawley rats received paired 2 × 2-cm burn wounds, which were subsequently inoculated with known virulence factor-producing methicillin-resistant Staphylococcus aureus or media alone on postinjury day 1. Infected animals received twice-daily tigecycline (high or low dose), twice-daily clindamycin (high or low dose), or saline alone (positive controls). Daily sampling and imaging assessments were performed. Results: Bacterial counts and toxin levels were reduced significantly in antibiotic-treated groups relative to positive controls (P < .001). Results from day 7 showed measurable toxin levels in clindamycin-treated, but not tigecycline-treated, wounds. Imaging analysis revealed a return of wound perfusion in tigecycline-treated animals similar to the sham animals. Transcript analysis using polymerase chain reaction and polymerase chain reaction arrays demonstrated downregulation of gene expression in antibiotic-treated animals as compared with positive controls. Conclusions: Overall, this study supports the use of tigecycline in the treatment of methicillin-resistant Staphylococcus aureus-infected burn wounds. While both protein synthesis inhibitors are effective, tigecycline appears to be superior in controlling toxin levels, enabling better wound healing.

Rheumatoid myositis leading to acute lower extremity compartment syndrome: a case-based review.

Muscle pain and weakness in a rheumatoid arthritis (RA) patient has a broad differential, and myositis should be considered early in the disease course as serious limb and life-threatening sequelae may occur. A 55-year-old woman with a past medical history of methotrexate-controlled RA presented with right leg pain for 4 days. The patient suffered sensory loss in the right foot and decreased strength in the toes. Lab tests revealed elevated creatine kinase, ESR, and anti-rheumatoid factor antibody titers. CT scan revealed myositis of posterior compartment muscles. Progressive edema, pain, and neuromuscular deficits persisted despite steroid and antibiotic therapy, so the patient was taken for urgent fasciotomy for acute compartment syndrome. The muscle biopsy showed diffuse mononuclear cell infiltration as well as perivascular and perineural involvement consistent with rheumatoid myositis (RM). The patient did well post-op on a prednisone taper. This case underlines the systemic nature of RA and exemplifies the severity of inflammation that may lead to grave consequences such as compartment syndrome. The histopathology is diagnostic when there is evidence of mononuclear cell infiltration; however, this is not entirely specific. Early, aggressive therapy with immunosuppressives is warranted in such patients. RM has not, to our knowledge, been recorded to cause acute compartment syndrome. Clinicians should be aware of this uncommon manifestation of RA keeping the various presentations of rheumatoid disease in mind when faced with these patients.

Regional neurovascular inflammation and apoptosis are detected after electrical contact injury.

High-voltage electrical injuries are a devastating form of trauma often treated in burn centers. Examining superficial wounds alone may lead to an inaccurate assessment of local, regional, and systemic severity of injury. In this work, the neurovasculature at sites regionally distinct from the contact wound were assessed for cellular pathology. Nine male Sprague-Dawley rats subjected to 1000 V direct-current shocks were separated into three groups: high-shock (>10-second contact), low-shock (<4-second contact), and control. Injury video was captured with a forward-looking infrared camera, and a thermal excitation analysis was performed. The neurovascular bundles from the iliofemoral region to the distal posterior tibial region were dissected from the hind limbs of the shocked animals and stained by immunohistochemistry for antibodies specific to apoptosis (APO) 1, caspase-3, activating transcription factor 3, high-mobility group box-1, granulocyte-macrophage colony-stimulating factor and interleukin-6. Real-time reverse-transcription polymerase chain reaction was used to quantify differential transcript levels of superoxide dismutases 1, 2, and 3 and heat-shock protein 70 from peripheral blood mononuclear cells and liver tissue. Finally, a protein array was used to identify key inflammatory cytokines in blood plasma. Significant dose-dependent trends were identified in apoptotic markers as well as inflammatory markers in both arterial and nerve tissues. Although arterial tissue exhibited a gradual decline in these markers proximally from the wound site, nerve tissue maintained a constant level at every location. Transcript analysis revealed an up-regulation of extracellular superoxide dismutase, and down-regulation of heat-shock protein 70, whereas plasma inflammatory cytokine levels indicated no significant changes. Thermal excitation analysis revealed a linear temperature increase, with a dose-dependent thermal maximum. In this study the authors have shown that neurovascular APO and inflammation are present at locations extremely proximal to electrical injury contact sites and this appears to be dose-dependent. Nerve tissue APO and inflammation may extend farther proximally than the iliofemoral region, and multiple proapoptotic mechanisms may be activated. No systemic inflammatory response was indicated in this study.

Treatment with an oxazolidinone antibiotic inhibits toxic shock syndrome toxin-1 production in MRSA-infected burn wounds.

Mortality rates in burn patients increase if they experience complications of infection. Frequently, the organisms associated with such infections are Staphylococci, including antibiotic-resistant species such as methicillin-resistant Staphylococcus aureus. Virulence factor production can further complicate treatment as a localized toxin presence may derail the healing process and allow a more invasive infection, while a toxin that becomes systemic can induce shock and cause host immune disruption. Male rats were anesthetized and subjected to full-thickness burn wounds. One day postinjury, wounds were inoculated with Toxic Shock Syndrome Toxin-1-producing methicillin-resistant S. aureus. Animals were then divided into three treatment groups: vancomycin, linezolid, or positive control. For nine additional days, animals received twice-daily antibiotics and wound assessments, blood draws, and wound biopsies were performed. All animals had wound quantitative cultures that exceeded 1 × 10 colony forming units (CFU) per gram 1 day after inoculation. Linezolid treatment significantly reduced the bacterial counts in the wounds. Positive controls and vancomycin-treated animals had toxins in their wounds by day 5 and this remained throughout the study (ranging from 20-80 ng/ml). Linezolid-treated animals had significant decrease in toxin production (< 5 ng/ml), and in most cases toxins were undetectable. No animals became systemically infected with bacteria at any point during the study. Superantigen production in burn wounds has morbid consequences in terms of long-term wound healing. A S. aureus burn wound infection model was created that allowed the study of the effect of two standard-use antibiotics on local burn wound pathophysiology. Most noteworthy is that low-dose linezolid arrested toxin production in the wound.

Examination of local and systemic in vivo responses to electrical injury using an electrical burn delivery system.

Electrical injuries are devastating and are difficult to manage due to the complexity of the tissue damage and physiological impacts. A paucity of literature exists which describes models for electrical injury. To date, those models have been used primarily to demonstrate thermal and morphological effects at the points of contact. Creating a more representative model for human injury and further elucidating the physics and pathophysiology of this unique form of tissue injury could be helpful in designing stage-appropriate therapy and improving limb salvage. An electrical burn delivery system was developed to accurately and reliably deliver electrical current at varying exposure times. A series of Sprague-Dawley rats were anesthetized and subjected to injury with 1000 V of direct current at incremental exposure times (2-20 seconds). Whole blood and plasma were obtained immediately before shock, immediately postinjury, and then hourly for 3 hours. Laser Doppler images of tissue adjacent to the entrance and exit wounds were obtained at the outlined time points to provide information on tissue perfusion. The electrical exposure was nonlethal in all animals. The size and the depth of contact injury increased in proportion to the exposure times and were reproducible. Skin adjacent to injury (both entrance and exit sites) exhibited marked edema within 30 minutes. In adjacent skin of upper extremity wounds, mean perfusion units increased immediately postinjury and then gradually decreased in proportion to the severity of the injuries. In the lower extremity, this phenomenon was only observed for short contact times, while longer contact times had marked malperfusion throughout. In the plasma, interleukin-10 and vascular endothelial growth factor levels were found to be augmented by injury. Systemic transcriptome analysis revealed promising information about signal networks involved in dermatological, connective tissue, and neurological pathophysiological processes. A reliable and reproducible in vivo model has been developed for characterizing the pathophysiology of high-tension electrical injury. Changes in perfusion were observed near and between entrance and exit wounds that appear consistent with injury severity. Further studies are underway to correlate differential mRNA expression with injury severity.

Neuroendocrine differentiation in extrahepatic bile duct carcinomas and its prognostic significance.

Neuroendocrine differentiation is known to be one of the prognostic factors in many carcinomas. However, the characteristics of neuroendocrine differentiation are not well elucidated in extrahepatic bile duct (EBD) carcinomas. One hundred ninety-four cases of EBD carcinomas were analyzed using immunohistochemistry with synaptophysin and chromogranin. The tumors were graded as degree 0, 1, and 2 when the positive tumor cells were 5% or less, 6% to 25%, and 26% or more, respectively. Immunohistochemical results were compared with clinicopathologic variables and survival rate. Synaptophysin and chromogranin were positive in 54 (27.8%) and 74 (38.1%) cases, respectively. Thirty-four cases (17.5%) were positive for both synaptophysin and chromogranin, 20 (10.3%) and 40 cases (20.6%) were positive only for synaptophysin and for chromogranin, respectively, and 100 cases (51.6%) were negative for both markers. There was a significant survival difference between overall synaptophysin-positive (median, 27 months) and synaptophysin-negative (38 months) groups (P < .05). However, there was no survival difference between chromogranin-positive and chromogranin-negative groups. There was a significant survival difference between the dual-positive expression to synaptophysin and chromogranin group (median, 21 months) and the dual-negative expression group (median, 35 months; P < .05). In summary, synaptophysin expression was an important prognostic factor because synaptophysin-positive cases showed a worse prognosis than synaptophysin-negative cases. The more tumor cells expressed chromogranin, the poorer the survival. Therefore, immunohistochemical studies for neuroendocrine differentiation may be helpful in routine pathological examinations for evaluating the survival and the prognosis of patients with EBD carcinomas.

Analysis of extrahepatic bile duct carcinomas according to the New American Joint Committee on Cancer staging system focused on tumor classification problems in 222 patients.

BACKGROUND: Although the sixth edition of the American Joint Committee on Cancer (AJCC) staging system for extrahepatic bile duct carcinoma was updated, the system has a problem on T classification due to its ambiguous definition of T1 as "tumor confined to bile duct histologically" and T2 as "tumor invading beyond the bile duct." METHODS: The authors considered the outermost part of the muscle layer or fibrous tissue as within the extrahepatic bile duct and considered the area starting from large clusters of adipose tissue as beyond the extrahepatic bile duct. After designing a precise definition of the extrahepatic bile duct wall, they analyzed the new AJCC staging system in 222 patients with of extrahepatic bile duct carcinomas. Then, other clinicopathologic variables for prognosis were evaluated using univariate and multivariate analyses. RESULTS: The 5-year survival rates for patients with tumors that were classified as T1, T2, T3, and T4 were 53.1%, 29.7%, 24.9%, and 0%, respectively. There was a significant difference in survival between patients with T1 tumors and T2 tumors (P < 0.05), but not between patients with T2 tumors and T3 tumors. Significant prognostic factors included depth of invasion (P < 0.005), lymph node metastasis (P < 0.005), and patient age (P < 0.05). CONCLUSIONS: Based on a proposed histologic definition, depth of invasion was practical for evaluating the prognosis of patients with middle and upper extrahepatic bile duct carcinomas. Therefore, the authors recommended changing the current pT1 and pT2 classifications to more precise pathologic terminology.

Superficial vs deep pancreatic parenchymal invasion in the extrahepatic bile duct carcinomas: a significant prognostic factor.

Pancreatic invasion of the extrahepatic bile duct (EBD) carcinomas is known to have a poor outcome. We hypothesized that EBD carcinoma showing shallow invasion to the pancreas may have a better outcome than the usual deep pancreatic invasion. We divided 87 cases of the distal EBD carcinomas into superficial and deep pancreatic invasion groups according to degrees of the pancreatic invasion. The superficial pancreatic invasion group included cases with tumor abutting the pancreatic lobule or pancreatic parenchymal invasion equal to or less than 1 mm from the uppermost portion of the pancreatic lobule or tumors invading into the fibroadipose tissue between pancreatic lobules without parenchymal invasion. The deep invasion group consisted of tumors with more than 1 mm pancreatic parenchymal invasion. The cases with superficial pancreatic invasion showed significantly better survival rate than those with deep pancreatic invasion (P<0.001). Therefore, we recommend that a specific remark on the pathology report about the presence or absence of parenchymal invasion and the depth of invasion of the pancreas is required for managing patients and determining the prognosis. We also recommend that the current pT3 stage of distal EBD carcinomas be subdivided into superficial (pT3a) and deep pancreatic invasion (pT3b).