Epidermal growth factor receptor regulates fibrinolytic pathway elements in cervical cancer: functional and prognostic implications.

Epidermal growth factor receptor (EGFR) signaling and components of the fibrinolytic system, including urokinase-type plasminogen activator (uPA) and thrombomodulin (TM), have been implicated in tumor progression. In the present study, we employed cBioPortal platform (http://www.cbioportal.org/), cancer cell lines, and an in vivo model of immunocompromised mice to evaluate a possible cooperation between EGFR signaling, uPA, and TM expression/function in the context of cervical cancer. cBioPortal analysis revealed that EGFR, uPA, and TM are positively correlated in tumor samples of cervical cancer patients, showing a negative prognostic impact. Aggressive human cervical cancer cells (CASKI) presented higher gene expression levels of EGFR, uPA, and TM compared to its less aggressive counterpart (C-33A cells). EGFR induces uPA expression in CASKI cells through both PI3K-Akt and MEK1/2-ERK1/2 downstream effectors, whereas TM expression induced by EGFR was dependent on PI3K/Akt signaling alone. uPA induced cell-morphology modifications and cell migration in an EGFR-dependent and -independent manner, respectively. Finally, treatment with cetuximab reduced in vivo CASKI xenografted-tumor growth in nude mice, and decreased intratumoral uPA expression, while TM expression was unaltered. In conclusion, we showed that EGFR signaling regulated expression of the fibrinolytic system component uPA in both in vitro and in vivo settings, while uPA also participated in cell-morphology modifications and migration in a human cervical cancer model.

Epidermal growth factor receptor regulates fibrinolytic pathway elements in cervical cancer: functional and prognostic implications

Epidermal growth factor receptor (EGFR) signaling and components of the fibrinolytic system, including urokinase-type plasminogen activator (uPA) and thrombomodulin (TM), have been implicated in tumor progression. In the present study, we employed cBioPortal platform (http://www.cbioportal.org/), cancer cell lines, and an in vivo model of immunocompromised mice to evaluate a possible cooperation between EGFR signaling, uPA, and TM expression/function in the context of cervical cancer. cBioPortal analysis revealed that EGFR, uPA, and TM are positively correlated in tumor samples of cervical cancer patients, showing a negative prognostic impact. Aggressive human cervical cancer cells (CASKI) presented higher gene expression levels of EGFR, uPA, and TM compared to its less aggressive counterpart (C-33A cells). EGFR induces uPA expression in CASKI cells through both PI3K-Akt and MEK1/2-ERK1/2 downstream effectors, whereas TM expression induced by EGFR was dependent on PI3K/Akt signaling alone. uPA induced cell-morphology modifications and cell migration in an EGFR-dependent and -independent manner, respectively. Finally, treatment with cetuximab reduced in vivo CASKI xenografted-tumor growth in nude mice, and decreased intratumoral uPA expression, while TM expression was unaltered. In conclusion, we showed that EGFR signaling regulated expression of the fibrinolytic system component uPA in both in vitro and in vivo settings, while uPA also participated in cell-morphology modifications and migration in a human cervical cancer model.

Reduced opioid requirements following unicompartmental knee arthroplasty compared with total knee arthroplasty.

AIMS: Unicompartmental knee arthroplasty (UKA) provides improved early functional outcomes and less postoperative morbidity and pain compared with total knee arthroplasty (TKA). Opioid prescribing has increased in the last two decades, and recently states in the USA have developed online Prescription Drug Monitoring Programs to prevent overprescribing of controlled substances. This study evaluates differences in opioid requirements between patients undergoing TKA and UKA. PATIENTS AND METHODS: We retrospectively reviewed 676 consecutive TKAs and 241 UKAs. Opioid prescriptions in morphine milligram equivalents (MMEs), sedatives, benzodiazepines, and stimulants were collected from State Controlled Substance Monitoring websites six months before and nine months after the initial procedures. Bivariate and multivariate analysis were performed for patients who had a second prescription and continued use. RESULTS: Patients undergoing UKA had a second opioid prescription filled 50.2% of the time, compared with 60.5% for TKA (p = 0.006). After controlling for potential confounders, patients undergoing UKA were still less likely to require a second prescription than those undergoing TKA (adjusted odds ratio (OR) 0.58, 95% confidence interval (CI) 0.42 to 0.81; p = 0.001). Continued opioid use requiring more than five prescriptions occurred in 13.7% of those undergoing TKA and 5.8% for those undergoing UKA (p = 0.001), and was also reduced in UKA patients compared with TKA patients (adjusted OR 0.33, 95% CI 0.16 to 0.67; p = 0.022) in multivariate analysis. The continued use of opioids after six months was 11.8% in those undergoing TKA and 8.3% in those undergoing UKA (p = 0.149). The multivariate models for second prescriptions, continued use with more than five, and continued use beyond six months yielded concordance scores of 0.70, 0.86, and 0.83, respectively. CONCLUSION: Compared with TKA, patients undergoing UKA are less likely to require a second opioid prescription and use significantly fewer opioid prescriptions. Thus, orthopaedic surgeons should adjust their patterns of prescription and educate patients about the reduced expected analgesic requirements after UKA compared with TKA. Cite this article: Bone Joint J 2019;101-B(7 Supple C):22-27.

Onlay foreskin flap anastomosed directly to the tunica albuginea: a short-term experimental study in rabbits.

INTRODUCTION: In severe hypospadias, urethral plate division is necessary for curvature correction. To configure the new urethra, an approach has been described using a foreskin flap directly anastomosed in an 'onlay' fashion to the tunica albuginea of the corpora cavernosa. Results suggest that it is possible to use the corpus cavernosum albuginea as the posterior wall of the neourethra without the need of a dorsal graft. OBJECTIVE: The present experimental study aimed to evaluate the histological characteristics and healing pattern of this procedure. STUDY DESIGN: Sixteen New Zealand male rabbits were divided into two groups of eight animals. Eight animals underwent 1-cm longitudinal dorsal incision of the penile urethra and the edges were anastomosed to the tunica albuginea (Group 1). Eight other animals underwent complete excision of 1.0 cm of penile urethra. Urethroplasty was performed using a foreskin flap directly anastomosed as an onlay to the albuginea, as shown in the figure (Group 2). Sacrifice and histological assessment was performed 2, 4, 8 and 12 weeks postoperatively. RESULTS: In Group 1, a mild inflammatory process was noted that became almost imperceptible at 12 weeks. Fibrosis was mild at all stages in this group. Over time, a regenerative epithelium covered the corpus cavernosum. Immunohistochemistry using specific CK-7 and CK-20 confirmed the presence of urothelium. No complications were microscopically detected in this group. Group 2 presented with a more intense inflammatory infiltrate, which also resolved over time. Fibrosis was slightly more intense in this group, especially in animals that had urethral strictures. Group 2 presented with three fistulas, two were associated with urethral stricture. Histological evaluation showed the presence of epithelization over the albuginea, which turned out to be similar to the normal urothelium over time and was confirmed by immunohistochemistry. Non-keratinized stratified squamous epithelium of the foreskin flap showed good integration to the urethra. DISCUSSION: Microscopic analysis showed that inflammation, fibrosis and complications were similar to previous studies. At 12 weeks there was a well-developed epithelium similar to normal urethra, which was confirmed by immunohistochemistry; this was similar to what occurs in the TIP technique, as previously demonstrated. It was hypothesized that the epithelium regeneration developed from the urethral edges, as demonstrated in other experimental studies. CONCLUSION: The albuginea was covered by mature urothelium after 12 weeks, which presumably grew from the urethral edges. The foreskin flap onlay that was directly anastomosed to the albuginea completely integrated and constituted the roof of the neourethra.

Reagent-free determination of amikacin content in amikacin sulfate injections by FTIR derivative spectroscopy in a continuous flow system / 药物分析学报

The quantitative estimation of amikacin (AMK) in AMK sulfate injection samples is reported using FTIR-derivative spectrometric method in a continuous flow system. Fourier transform of mid-IR spectra were recorded without any sample pretreatment. A good linear calibration (r40.999, %RSDo 2.0) in the range of 7.7-77.0 mg/mL was found. The results showed a good correlation with the manufacturer's and overall they all fell within acceptable limits of most pharmacopoeial monographs on AMK sulfate.

First Report of Guignardia psidii, an Ascigerous State of Phyllosticta psidiicola, Causing Fruit Rot on Guava in Venezuela.

During August 2003, guava fruit (Psidium guajava L.) cv. Red Dominicana from Cojedes state in Venezuela showed circular, purple-to-brown lesions (0.5 to 1.0 cm) that spread over all surfaces and became black and shrunken on severely affected fruit. Symptomatic tissues were plated aseptically on potato dextrose agar (PDA). Colonies that were initially gray and turned black with age were consistently isolated. The fungus was characterized by dense, submerged, brown-to-black mycelium with septate hyphae. Ascocarps were perithecial, abundant, granulose, subglobose to cylindric obpyriform, solitary or aggregated, mostly unilocular with prominent long necks; ascocarp walls were stromatic, composed of several layers of cells, thick walled, and deeply pigmented on the outside. Asci were subclavate to cylindrical, stipitate, 44 to 84 × 7 to 9 µm, and eight-spored; asci walls were thick and bitunicate. Ascospores were unicellular, hyaline, guttulate, fusiform ellipsoid, widest in the mid-region with rounded ends and gelatinous plugs, and 12 to 17 × 4.5 µm. Conidiomata were pycnidial, intermixed among ascocarps, variable in shape, dark brown, solitary or aggregated, ostiolate, and with long necks up to 1 mm. Pycnidial walls were pseudoparenchymatic, multicellular, and composed of many layers of brown compressed cells. Conidiogenous cells were hyaline, subglobose to cylindrical, and smooth, and holoblastic. Conidia were hyaline, unicellular, obovate, 6 to 12 (7.5) × 5 to 8 µm, slightly truncate at the bases, rounded at apices, guttulate, and provided a gelatinous envelope and apical appendage. Appendages were hyaline, tubular, smooth, and 3.0 to 4.5 × 0.5 µm. The fungus is homothallic because single ascospores and single conidia developed ascigerous states. The ascigerous state was identified as Guignardia psidii (1) and the anamorph as Phyllosticta psidiicola (1,2). Pathogenicity tests were conducted on detached fruits inoculated with monosporic cultures. Pathogenesis and symptom development only occurred when a mixture of mycelium, ascospores, and conidia was used as inoculum. The fungus was reisolated from symptomatic fruit tissues. To our knowledge, this is the first report of Guignardia psidii, an ascigerous state of Phyllosticta psidiicola from guava fruits in Venezuela. References: (1) B. A. Ullasa and R. D. Rawal. Curr. Sci. 53:435, 1984. (2) H. A. van der Aa. Page 95 in: No. 5, Stud. Mycol., 1973.

The yeast THO complex and mRNA export factors link RNA metabolism with transcription and genome instability.

The THO complex is a multimeric factor containing four polypeptides, Tho2, Hpr1, Mft1 and Thp2. Mutations in any of the genes encoding THO confer impairment of transcription and a transcription-dependent hyper-recombination phenotype, suggesting that THO has a functional role in gene expression. Using an in vivo assay developed to study expression of long and G+C-rich DNA sequences, we have isolated SUB2, a gene involved in mRNA splicing and export, as a multicopy suppressor of the gene expression defect of hpr1 Delta. Further investigation of a putative functional relationship between mRNA metabolism and THO revealed that mRNA export mutants sub2, yra1, mex67 and mtr2 have similar defective transcription and hyper-recombination phenotypes as THO mutants. In addition, THO becomes essential in cells with a defective Mex67 mRNA export er. Finally, we have shown that THO has the ability to associate with RNA and DNA in vitro. These results indicate a functional link between the processes of elongation and metabolism of nascent mRNA mediated by THO and mRNA export proteins, which have important consequences for the maintenance of genome stability.

Intermediate or chronic cutaneous leishmaniasis: leukocyte immunophenotypes and cytokine characterisation of the lesion.

The American cutaneous forms of leishmaniasis include immune-responder individuals with localised cutaneous leishmaniasis (LCL) and non-responder individuals with diffuse cutaneous leishmaniasis (DCL). Patients with intermediate or chronic cutaneous leishmaniasis (ICL) have increased morbidity due to the length of their illness, atypical forms and areas of compromise. In the present study, we evaluated the expression of the leukocyte antigens (CD4, CD8, CLA: cutaneous lymphocyte antigen, CD69, CD83 and CD1a) and cytokines (IFN-gamma, IL-4, IL-10 and TGF-beta 1) in the lesions of patients with ICL (n = 18) using an immunocytochemical procedure. ICL results were compared with the information for LCL (n = 19) and DCL (n = 4). The numbers of CD4+ and CD8+ T cells in ICL were similar to those of LCL lesions, but significantly different (P < or = 0.05) from DCL lesions. LCL lesions have about half the numbers of early activated CD69+ cells as ICL, but most are CLA+ skin homing memory T cells, whereas ICL lesions have the highest number of CD69+ T cells, but about one-third of these cells expressed CLA. This suggests that the granuloma of ICL patients contains many activated T cells that are unprimed to cutaneous-launched antigens, thus contributing to an aberrant immune response. In contrast, DCL granulomas presented the lowest numbers of activated CD69+ and CLA+ cells, associated with the characteristic tolerogenic state of these patients. The immunolocalisation of cytokines showed a mixed cytokine pattern in ICL lesions with many positive cells for IL-10, TGF-beta 1, IL-4 and IFN-gamma, with a preponderance of the first two, and different from the prevalent Th1 and Th2 responses associated with LCL and DCL lesions, respectively. CD1a+ Langerhans cells were decreased (P < or = 0.05) in both ICL (271 +/- 15 cells/mm2) and DCL (245 +/- 19 cells/mm2) as compared to LCL (527 +/- 54 cells/mm2) epidermis. The percentage of IL-10+ epidermal Langerhans cells in ICL (33.69), from the total CD1a+ population, was higher than in LCL (17.45). In addition, fewer CD83+ primed Langerhans cells were present in ICL epidermis. The diminished participation of epidermal Langerhans cells, causing a defective signalling by the epidermis, in ICL lesions may account for the tissue-damaging state observed in these patients.

A protein complex containing Tho2, Hpr1, Mft1 and a novel protein, Thp2, connects transcription elongation with mitotic recombination in Saccharomyces cerevisiae.

Transcription-induced recombination has been reported in all organisms from bacteria to mammals. We have shown previously that the yeast genes HPR1 and THO2 may be keys to the understanding of transcription-associated recombination, as they both affect transcription elongation and hyper-recombination in a concerted manner. Using a yeast strain that has the wild-type THO2 gene replaced by one encoding a His(6)-HA-tagged version, we have isolated an oligomeric complex containing four proteins: Tho2, Hpr1, Mft1 and a novel protein that we have named Thp2. We have reciprocally identified a complex containing Hpr1, Tho2 and Mft1 using anti-Mft1 antibodies in immunoprecipitation experiments. The protein complex is mainly nuclear; therefore, Tho2 and Hpr1 are physically associated. Like hpr1Delta and tho2Delta cells, mft1Delta and thp2Delta cells show mitotic hyper- recombination and impaired transcription elongation, in particular, through the bacterial lacZ sequence. Hyper-recombination conferred by mft1Delta and thp2Delta is only observed in DNA regions under transcription conditions. We propose that this protein complex acts as a functional unit connecting transcription elongation with the incidence of mitotic recombination.

Adhesion molecules in lesions of American cutaneous leishmaniasis.

Accessory signals, which include adhesion molecules, MHC-II molecules and cytokines, are necessary to foster the interaction between memory T cells and epidermal cells, that is required to promote cutaneous inflammatory responses. American cutaneous leishmaniasis (ACL) is characterized by a spectrum of immunological manifestations, and is a prototype disease for the study of regulatory mechanisms involved in immune protection against protozoal infection. In the present study, we show that diffuse cutaneous leishmaniasis (DCL) epidermis contains keratinocytes that do not express ICAM-1 and HLA-DR molecules. Langerhans cells (LC) are within normal values or somewhat lower, and a very few cells expressing the HB15 molecule--a new described member of the Ig superfamily--are found in such lesions. Mucocutaneous leishmaniasis (MCL) epithelium shows an increased expression of ICAM-1 and HLA-DR molecules, few HB15+ cells, and an absence of epithelial LC. Localized cutaneous leishmaniasis (LCL) epidermis displays ICAM-1+ keratinocytes organized in patches, a uniform expression of HLA-DR, hyperplasia of LC, and numerous HB15+ cells. In all forms of the disease, infiltrating T cells express more LFA-1 beta than LFA-1 alpha, but LFA-1 beta+ T cells are more abundant in LCL granulomas. In contrast, there are more LFA-1 alpha+ T cells in DCL and MCL than in LCL granulomas. LCL lesions also show the highest numbers of HB15+ cells within the granuloma. These results indicate the importance of adhesion molecules in ACL lesions, and open new possibilities for therapeutic schemes oriented towards the control of cell migration.

The clinical and immunological spectrum of American cutaneous leishmaniasis.

American cutaneous leishmaniasis is characterized by a spectrum of clinical manifestations. These include localized, often self-healing single lesions, intermediate forms which frequently produce mucosal lesions and often show exaggerated delayed-type hypersensitivity (DTH), and the rare diffuse cutaneous leishmaniasis in which no reaction of protective cell-mediated immunity or DTH can be demonstrated. Clinical, pathological and immunological studies have begun to unravel some of the mechanisms associated with different disease manifestations, dependent on complex interactions between the host immune response, measured in terms of indices including lymphocyte subsets and lymphokines in vitro and within active lesions, and different species of Leishmania.

Immunotherapy of localized, intermediate, and diffuse forms of American cutaneous leishmaniasis.

The clinical efficacy of immunotherapy for localized American cutaneous leishmaniasis with a combination of heat-killed Leishmania mexicana amazonensis promastigotes and viable BCG (bacille Calmette Guérin) has been compared with meglumine antimoniate chemotherapy and with BCG alone in a controlled clinical study in 217 patients. The results in the first two groups were comparable, with greater than 90% clinical cures with an average time of 16-18 w required for healing. The cure rate was considerably lower (42%) and more prolonged in the group receiving BCG alone. Secondary effects were observed in less than 5% of the patients receiving combined immunotherapy or BCG alone. In contrast, 49% of the patients receiving chemotherapy showed side effects. High therapeutic efficacy was also observed using combined immunotherapy in patients with intermediate and diffuse cutaneous leishmaniasis who were previously unresponsive to chemotherapy. Cure or clinical improvement was seen in all 11 patients with intermediate forms of the disease, and marked clinical improvement was observed in 9 of 10 patients with diffuse disease. The results on the efficacy of the combined vaccine in immunotherapy for American cutaneous leishmaniasis provide a strong rationale for studying its effectiveness in prophylactic trials.

Cell-mediated immunity in localized cutaneous leishmaniasis patients before and after treatment with immunotherapy or chemotherapy.

In previous studies of the treatment of localized cutaneous leishmaniasis (LCL) we demonstrated that the therapeutic efficiency of immunotherapy (BCG plus promastigotes of Leishmania mexicana) is equal to that of chemotherapy (Glucantime), without causing the serious side-effects of the drug treatment. In the present study, various aspects of cell-mediated immunity, including the lymphoproliferative response, and leucocyte subpopulations were evaluated both before treatment and after cure in 39 LCL patients who had received immunotherapy (IT), in 34 submitted to chemotherapy (CT), and in 14 patients cured by the administration of BCG alone. We demonstrated evident signs of T-cell activation in cured patients who had received either CT or IT. For example, an increased expression of IL-2 receptors was observed in such patients, compared to their pretreatment values. Also, a significant percentage of patients showed augmented in-vitro responses to mitogen, and both in-vitro and in-vivo reactivity to leishmanial antigen. No evidence was found for the development of an exaggerated immune response to Leishmania parasites in the IT group, because the final level of immunological reactivity was comparable to the CT group. Neither was there any difference in terms of the final immune response between the patients cured by BCG treatment alone and the other groups. However, the therapeutic efficiency of BCG was lower and the mean cure time was longer. We conclude that IT is very useful in the treatment of LCL patients because of its high efficiency, low propensity to produce side-effects, and the fact that it does not induce a state of hyper-reactivity.

Situación actual en inmunoterapia de Leishmania / Current situation in inmunotherapy of the Leishmaniasis

En un ensayo clínico controlado, con evaluación a ciegas y asignación al azar, se ha evaluado la eficacia terapéutica de una combinación de B.C.G. con promastigotes de L.Mexicana amazonensis muertos por calor (Inmunoterapia) en comparación con Antimoniaco de Meglumine (Quimioterapia) y un tercer grupo vacunado con B.C.G. solamente, con un total de 171 pacientes de Leishmaniasis Cutánea localizada (99 Inmunoterapia, 46 Quimioterapia y 26 con BCG). Los resultados obtenidos son comparables en los dos primeros grupos, tanto en el porcentaje de curación (más del 95% a las 32 semanas) como en el tiempo promedio de curación (alrededor de 17 semanas, en cambio en el grupo con BCG solamente los porcentajes de curación son muy bajos (38,5% a las 32 semanas) y el tiempo promedio de curación mucho más prolongado (26 semanas). Los efectos secundarios fueron leves e infrecuentes (menos del 5%) en inmunoterapia y con BCG solo, pero muy frecuente (48%) y severos en quimioterapia. Resultados preliminares indican así mismo buena eficacia terapéutica de la inmunoterapia en pacientes cutáneos y mucosos del área intermedia del espectro clínico-inmunológico y en pacientes con L.Cutánea Difusa (LCD). en este trabajo se discuten los fundamentos de la inmunoterapia en Leishmaniasis en base al espectro de deficiencias de inmunidad celular y se plantean las perspectivas de la inmunoprofilaxis en base a estos conceptos

Demonstration of an indomethacin-sensitive mechanism regulating immune reactivity in American cutaneous leishmaniasis patients.

We investigated some aspects of the regulation of the immune response that were sensitive to the effect of indomethacin (INDO), an inhibitor of prostaglandin synthesis, in 84 patients with American cutaneous leishmaniasis (ACL), and in normal controls. The patients were classified on the basis of clinical and histopathological criteria as suffering localized (LCL), mucocutaneous (MCL) or diffuse (DCL) forms of the disease. The responses in vitro to mitogens (PHA and Con A) and leishmanial antigens were evaluated in the presence or absence of INDO. It was found that the drug significantly increased in vitro the mitogenic stimulation by PHA of peripheral blood mononuclear cells from LCL, MCL and DCL patients, but the effect was less evident in the controls. Considering specific responses to leishmanial antigens, we showed that in the presence of INDO, these were significantly increased in LCL patients, but not in MCL or DCL. Also, only in LCL was an inverse correlation found between the initial response to leishmanial antigen and the increase caused by INDO. Significant correlations were found between the INDO-induced enhancement of PHA and Con A responses in the patient groups, but not in the controls. In LCL patients there was a significant correlation between the increases caused by INDO of the mitogen and antigen responses. It can be suggested that an indomethacin-sensitive (prostaglandin dependent) suppressor mechanism operates in LCL patients, that is possibly responsible for the modulation of the immune response against the parasite. In MCL, where this suppressive mechanism is apparently not functional, the response to the parasite is intense, and a possible consequence of this could be tissue damage. Our results indicate, however, that the anergy observed in DCL patients is not due to an involvement of prostaglandins in the suppression of the specific immune response.

Immunotherapy versus chemotherapy in localised cutaneous leishmaniasis.

In a randomised trial a combination vaccine consisting of live BCG together with killed leishmania promastigotes was compared with a standard antimonial regimen in 94 patients with localised cutaneous leishmaniasis. Three vaccinations over 32 weeks gave a similar cure rate (94%) to three 20-day courses of meglumine antimonate. In the immunotherapy group side-effects were few (5.8%) and slight whereas in the chemotherapy group they were frequent (52.4%) and often serious. Immunotherapy is a low-cost, low-risk alternative to chemotherapy in localised cutaneous leishmaniasis, applicable by primary health services in rural areas.

New projections in the development of a vaccination model using two microorganisms and its application in leishmaniasis and leprosy

Conventional vaccination is oriented toward the prevention of disease in individuals capable of developing normal immune responses. A new model of vaccination employing two microorganisms has been described for the correction of variable degrees of antigen-specifit deficiency in the development of effective cell-mediated immunity in two diseases, leprosy and cutaneous leishmaniasis, both of which are characterized by a spectrum of clinical manifestations. A schematic representation of the immunologic defect in the severe and progressive forms of these diseases and a possible mechanism for its correction using this vaccine model are presented. Immunotherapeutic and immunoprophylactic applications of the model are described, with particular reference to recent experience in the immunotherapy of localized cutaneous leishmaniasis. The efficacy, virtual absence of secondary effects, ease of administration and low cost of this therapeutic modality indicate that it offers an important option or field use in endemic of leishmaniasis