The Effects of Macrophages on the Survival of Random Skin Flaps in Swine

Tissue macrophages play a critical role in neovascularization by releasing angiogenic cytokines. Macrophages normally arrive into a wound bed 48 to 96 hours following an injury. ^1. Introducing macrophages into a wound bed at the time of closure would theoretically stimulate neovascularization in the traumatized tissue prior to what is normally observed. The ability to promote early angiogenesis could be an important factor in the survival of an extended skin flap. By taking advantage of advanced cell-sorting techniques, the authors developed the first study to evaluate the effect of placing a purified autologous macrophage population into an extended skin flap. We created 72 dorsally based random skin flaps in Yorkshire pigs; 48 of these wounds received autologous macrophages while 24 flaps served as controls. The macrophages were obtained by utilizing monoclonal antibodies in conjunction with flow cytometry. The skin flaps were evaluated on postoperative day 6 for their viability. Analysis of the data showed no statistically significant difference between the control and treatment flaps. There was, however, a trend of increased survival for flaps treated with macrophages. This is the first study to investigate using a purified population of cells to improve the survival of random skin flaps.     

Chylothorax

During a high-speed road traffic accident, a 26-year-old man suffered multiple fractures of his thoracic vertebrae and bilateral pneumothoraces. The day after admission and commencement of nasogastric feeding, milky fluid was noted in his right chest drain. Feeding was stopped and a contrast oesophogram and oesophagoscopy were performed, which were normal. The chylothorax quickly resolved and both drains were removed on day 6. Initial treatment of chylothorax aims to decompress the pleural space and minimise chyle production by not feeding the patient via the enteral route. Most authors recommend conservative management for 2 weeks or more unless certain parameters are met: average daily chyle loss of > 1.5 l for a 5-day period, or imminent nutritional complications. In this case, surgical management of the chylothorax and spinal fractures was planned. However, conservative management was successful, highlighting the fact that early aggressive surgical intervention for chyle leaks in blunt trauma is not necessary.     

Expression of PTEN protein in patients with laryngeal squamous cell carcinoma

OBJECTIVE: PTEN (phosphatase and tensin homologue deleted on chromosome 10), also referred to as MMAC1 (mutated in multiple advanced cancers) gene was recently identified as a putative tumor suppressor in a variety of malignant tumors. PTEN expression has been investigated in some squamous cell carcinomas (SCC) of head and neck. However, there is only little knowledge about laryngeal malignancies. Therefore, we examined PTEN product protein immunohistochemically in 30 consecutive laryngeal specimens from patients with laryngeal SCC and compared the results according to the clinicopathologic characteristics of the patients. METHOD: Surgical resection specimens of patients with laryngeal SCC were stained for PTEN protein using a primary rabbit polyclonal anti-PTEN antibody. Standard avidin-biotin immunohistochemical analysis was used to process the sections. The extent and intensity of PTEN staining in the specimens were compared according to the age and sex of the patients and localization, differentiation, size and stage of the tumor. RESULTS: Out of 30 tumoral specimens (23 glottic and 7 supraglottic) 22 showed decreased PTEN staining intensity compared to the adjacent normal tissue. The extent of cytoplasmic PTEN staining was significantly less in supraglottic tumors (p < 0.05). When characteristics of the patients were analyzed according to the extent of cytoplasmic PTEN staining no difference was observed according to age, sex, measure, differentiation, T or N status. CONCLUSION: A significant decrease in the extent of PTEN staining was observed in supraglottic SCC. It could be worthwhile to test if PTEN expression is diminished in patients with more aggressive laryngeal tumors, with special attention to tumor localization in larger series.     

A pilot study evaluating the efficacy and toxicity of biweekly gemcitabine and pegylated liposomal doxorubicin in recurrent platinum-resistant epithelial ovarian cancer

Background Both gemcitabine and pegylated liposomal doxorubicin (PLD) are antineoplastic drugs with clinical activity in patients with platinum-resistant ovarian cancer. The present study was designed to assess the efficacy and safety of biweekly scheduled gemcitabine and PLD combination therapy in such patients. Methods Eighteen women with ovarian cancer that had recurred within 6 months after standard carboplatin and paclitaxel therapy were eligible for enrollment. Gemcitabine 2000 mg/m² and PLD 20 mg/m² were administered intravenously on days 1 and 15 of a 28-day cycle. Results Hematological toxicity was mild. No severe (grade III/IV) leucopenia/neutropenia or thrombocytopenia was observed. Severe anemia was seen in only 3 (17%) patients. Several other severe nonhematological adverse effects were well tolerated and easily managed. The overall response rate was 28% (5 of 18; 95% confidence interval [CI], 10%–54%) with 2 (11%) complete and 3 (17%) partial responses. The median overall survival time was 17 months (range, 1 to 25 months). The median survival for patients with clinical benefit including disease response or stabilization was 17 months (range, 3 to 26 months) compared to that of patients with progressive disease, which was 2 months (range, 1 to 11 months; P = 0.04). Conclusion A biweekly schedule of gemcitabine combined with PLD is an active and safe chemotherapy regimen with acceptable and easily manageable toxicities in women with recurrent platinum-resistant ovarian cancer.     

Chemotherapy with pegylated liposomal doxorubicin and cisplatin in recurrent platinum-sensitive epithelial ovarian cancer

BACKGROUND: Pegylated liposomal doxorubicin (PLD) is the only nonplatinum agent to significantly improve survival in patients with platinum-sensitive recurrent ovarian cancer. The present study was designed to assess the efficacy and safety of PLD plus cisplatin combination therapy in these patients. METHODS: Twenty-two women (median age, 57 years) with ovarian cancer that recurred 6 months or more after standard carboplatin and paclitaxel therapy were eligible for enrollment. Cisplatin was administered intravenously as a 60-min infusion on day 1, at a single dose of 60 mg/m(2), and PLD was given intravenously as a 1-h infusion on day 2, at a dose of 50 mg/m(2). Treatment cycles were repeated on an outpatient basis every 28 days. RESULTS: Hematological toxicity was mainly leucopenia/neutropenia, and this was the principal dose-limiting toxicity. Severe (grade III-IV) leucopenia/neutropenia was observed in 7 (32%) and 9 (41%) patients, respectively. Only 2 (9%) patients were complicated by febrile neutropenia. Grade III-IV anemia occurred in only 4 (18%) patients. Severe thrombocytopenia was not noted; only 5 patients (23%) had grade I-II toxicity. NO toxicity in biochemical parameters was noted. Several severe nonhematological adverse effects were managed according to standard protocols and were transient, as well as being well-tolerated. Twenty-one patients were evaluated for response. The overall response rate was 62% (13 of 21; 95% confidence interval [CI], 38%-82%), with four (19%) complete and nine (43%) partial responses. At the time of last follow-up, all of the 22 patients were alive. The median follow-up period was 8.5 months (range, 2 to 22 months). CONCLUSION: PLD combined with cisplatin at the schedule and dosage used in this study is an active and safe second-line chemotherapy regimen with acceptable and easily manageable toxicities in women with platinum-sensitive recurrent ovarian cancer.     

Physical detection of influenza A epitopes identifies a stealth subset on human lung epithelium evading natural CD8 immunity

Vaccines eliciting immunity against influenza A viruses (IAVs) are currently antibody-based with hemagglutinin-directed antibody titer the only universally accepted immune correlate of protection. To investigate the disconnection between observed CD8 T-cell responses and immunity to IAV, we used a Poisson liquid chromatography data-independent acquisition MS method to physically detect PR8/34 (H1N1), X31 (H3N2), and Victoria/75 (H3N2) epitopes bound to HLA-A*02:01 on human epithelial cells following in vitro infection. Among 32 PR8 peptides (8–10mers) with predicted IC₅₀ < 60 nM, 9 were present, whereas 23 were absent. At 18 h postinfection, epitope copies per cell varied from a low of 0.5 for M1_3–11 to a high of >500 for M1_58–66 with PA, HA, PB1, PB2, and NA epitopes also detected. However, aside from M1_58–66, natural CD8 memory responses against conserved presented epitopes were either absent or only weakly observed by blood Elispot. Moreover, the functional avidities of the immunodominant M1_58–66/HLA-A*02:01-specific T cells were so poor as to be unable to effectively recognize infected human epithelium. Analysis of T-cell responses to primary PR8 infection in HLA-A*02:01 transgenic B6 mice underscores the poor avidity of T cells recognizing M1_58–66. By maintaining high levels of surface expression of this epitope on epithelial and dendritic cells, the virus exploits the combination of immunodominance and functional inadequacy to evade HLA-A*02:01-restricted T-cell immunity. A rational approach to CD8 vaccines must characterize processing and presentation of pathogen-derived epitopes as well as resultant immune responses. Correspondingly, vaccines may be directed against “stealth” epitopes, overriding viral chicanery.Human influenza is an acute respiratory infection caused by Orthomyxoviridae, a family of single-stranded, negative sense RNA viruses containing a segmented genome. The influenza virion is enveloped by a lipid bilayer derived from the host cell with hemagglutinin (HA), neuraminidase (NA), and matrix 2 (M2) transmembrane proteins of the virus exposed and antibody (Ab) accessible. HA is the most abundant protein on the surface and is the principle antigen recognized by humoral immunity (1). RNA replication is error prone, with influenza A virus (IAV) averaging roughly one error for each replicated genome (2). Notably, the HA protein manifests a high functional tolerance to sequence variation not evident in some of the internal proteins (3). Current vaccines protect largely by inducing Abs against the HA and NA proteins but require continual reformulation based on inferring the dominant strains that will circulate in the upcoming flu season, an imperfect science at best. Recognizing the challenges for universal protection against influenza via antibodies, there has been extensive discussion about vaccines harnessing cellular immunity (4–6). Cytotoxic T lymphocytes (CTLs), primarily CD8⁺ T cells, can recognize and kill infected cells when their T-cell receptors (TCRs) recognize fragments of viral proteins that are in turn bound to major histocompatibility complex (MHC in general; for humans, HLA) proteins on the surface of infected cells. These peptides can derive from segments of internal proteins conserved among IAV strains so that CTLs targeting these sequence-constrained peptides would remain effective as surface HA and NA antigens, in contrast, change by genetic drift or shift. There is substantial evidence for T-cell–mediated protection in mice (7, 8), but qualified evidence in humans (9, 10). The limited CTL protection has been ascribed to slow cellular responses against IAV, a latency reflecting the expansion and activation of central memory T cells in lymph nodes and the subsequent recruitment of CTLs to infected lung (11). The recent identification of CD8⁺ effector resident memory T cells (T_RM) within barrier tissues postinfection (12, 13) could challenge this picture. However, vaccines for cellular immunity are at an early stage of development and what formulations and delivery methods are needed to harness T_RM immunity is not well understood. In particular, the pathogen-derived peptides bound to HLA molecules on the surface of IAV-infected lung epithelial cells are unknown. As a T cell monitors a single MHC-restricted antigen, surveillance is constrained by the numbers of antigen-specific cells and their motility in lung parenchyma under homeostatic conditions. Sentinel T_RM cell populations lodged in barrier tissues are limited, so that inducing irrelevant specificities will necessarily displace those with useful specificities. In contrast, the fluid volume surrounding a cell at the site of infection can contain a large set of antibodies recognizing diverse antigens. Rational T-cell vaccine development must focus on the peptides directly presented by infected lung epithelium and identify those peptides that can be recognized by high avidity T_RM cells.IAV peptides displayed by infected cells are conventionally identified by T-cell functional assays. In principle this “reverse immunology” only identifies what epitopes the antigen-experienced host has recognized, not what can or should be recognized. As such, reverse immunology is a poor guide for vaccine development. Here new acquisition and analysis methods in mass spectrometry (MS) are applied to directly identify these peptides. New methodology is necessary as conventional data-dependent acquisition (DDA) MS, identifying ions largely in order of signal intensity, cannot plow deep enough into the sample to uncover the IAV peptides underneath an ocean of “self” peptides. Reflecting the prevalence of the HLA-A2 allele in the population and the large number of studies of IAV infection and CD8⁺ T-cell responses restricted by this allele, this study focuses on antigen presentation by HLA-A*02:01 (A2). Our results identify previously unrecognized IAV epitopes that may be useful in vaccine formulations, question the veracity of functionally ascribed epitope display, and show via stable isotope labeling by amino acids in culture (SILAC) that positive strand RNA-derived translation and not protein cross-presentation is the basis of A2 IAV peptidome array on dendritic cells (DCs) that phagocytose non-A2 IAV-infected UV-irradiated cells. In addition, our data rationalize why an immunodominant CD8 T-cell response to the highly conserved M1_58–66 peptide GILGFVFTL does not provide sterilizing immunity to IAV.     

Comparison of the beneficial effect of melatonin on recovery after cut and crush sciatic nerve injury: a combined study using functional, electrophysiological, biochemical, and electron microscopic analyses

Purpose

Following tissue injury, melatonin is known to reduce detrimental effects of free radicals by stimulating antioxidant enzymes and also to inhibit posttraumatic polymorphonuclear infiltration. Beneficial effects after peripheral nerve injury have been suggested, but not studied in detail. Therefore, we aimed to elucidate the effects of melatonin on the recovery of the lesioned rat sciatic nerve by means of combined analysis.

Methods

A total number of 90 rats were randomly distributed into six groups: control (group 1), sham-operated (group 2), sciatic nerve cut (group 3), sciatic nerve cut + melatonin treatment (group 4), sciatic nerve crush (group 5), and sciatic nerve crush + melatonin treatment (group 6). Melatonin was administered intraperitoneally at a dose of 50 mg/kg/day for 6 weeks. Recovery of function was analyzed by assessment of the sciatic functional index based on walking track analysis, somatosensory evoked potentials, biochemical quantification of malondialdehyde, antioxidant enzymes levels, and ultrastructural analysis.

Results

Our data showed the beneficial effect of melatonin on sciatic nerve recovery. Rats treated with melatonin demonstrated better structural preservation of the myelin sheaths compared to the nontreated group. The biochemical analysis confirmed the beneficial effects of melatonin displaying lower lipid peroxidation and higher superoxide dismutase, catalase, and glutathione peroxidase activities in sciatic nerve samples in comparison to nontreated groups.

Conclusions

The beneficial effects of melatonin administration on the recovery of the cut and crush injured sciatic nerve may be attributed to its antioxidant properties. Based on these investigations, we think that our data would be helpful for clinicians who deal with peripheral nerve injuries.