In vivo and in vitro activity of venom from the endoparasitic wasp Pimpla turionellae (L.) (Hymenoptera: Ichneumonidae)

The biological activity of venom from Pimpla turionellae L. (Hymenoptera: Ichneumonidae) was examined in vivo toward larvae and pupae of Galleriae mellonella L. (Lepidoptera: Pyralidae), and in vitro toward bacterial and fungal cultures, as well as cultured insect cells. Pupae of G. mellonella were far more susceptible to the venom than larvae. At low doses of venom [0.1 venom reservoir equivalents (VRE)], pupal abdominal mobility was inhibited within 30 min, and by 24 h, all pupae injected with venom concentrations >0.5 VRE were completely paralyzed. These same doses of venom resulted in an inhibition of adult emergence. Host larvae were far less sensitive to wasp venom as evidenced by all venom injected larvae remaining responsive to mechanical stimulation by 1 h post injection, even at concentrations equivalent to 1 venom reservoir. Eventually (>2 h at 25 degrees C), venom-injected larvae became immobile, then flaccid, and all died within 24 h post-injection. At lower concentrations of wasp venom, the onset of paralysis was delayed by comparison to that evoked by 1 VRE, and few host larvae were able to pupate. Development of host larvae to adult emergence was also reduced in a dose-dependent manner, with eclosion completely prevented at high concentrations (>0.5 VRE) of venom. Venom doses <0.5 VRE did not appear to induce paralysis or alter larval development. When venom was incubated with bacterial or fungal cultures, no antimicrobial activity was detected. However, wasp venom was found to be cytotoxic and cytolytic to cultured cells derived from the cabbage looper Trichoplusia ni Hubner (Lepidoptera: Noctuidae) and the yellow fever mosquito, Aedes aegypti (L.) (Diptera: Culcidae). Though both cell types displayed similar susceptibility in terms of LC50s, the lepidopteran cells responded much more rapidly with regard to the onset of morphological changes and the timing of cell death. A possible mode of action for the venom is discussed.     

Emergence of regulatory CD4+ T cell response to repetitive stimulation with antigen-presenting cells in vitro: implications in designing antigen-presenting cell-based tumor vaccines

Because APCs play a crucial role in the generation of T cell-mediated immune responses, numerous clinical trials with APC-based vaccines have been initiated in different types of human cancers. Encouraging results have emerged from some of these initial studies. Thus far, APC-based vaccinations usually include multiple rounds of immunization. With this approach, although we and others have detected induction of Ag-specific CTL responses in vaccinated patients after stimulation with the same APC-based immunogen, in vitro we also find that repetitive in vitro stimulation with Ag-loaded APC can, at times, lead to the emergence of noncytolytic CD4+ T cells exhibiting the characteristic phenotype of Th2 cells. These noncytolytic CD4+ T cells synthesize large quantities of type 2 cytokines such as IL-4 and IL-10 on stimulation with the autologous APC or tumor cells in an MHC class II-restricted manner. Further, these CD4+ T cells and a cell-free supernatant factor block the activation of fresh T lymphocytes. The supernatant factor also exhibits a marked inhibitory effect on the expression of the costimulatory molecules, CD80 and CD86, by APC. The inhibitory effect of the supernatant factor can be abrogated by neutralizing IL-10 in the supernatant. These observations therefore have implications in the APC-based tumor vaccine protocol design.     

Immunization with a tumor-cell-lysate-loaded autologous-antigen-presenting-cell-based vaccine in melanoma

The discoveries of human melanoma-associated antigens in molecular terms have renewed interest in peptide- or peptide- and antigen-presenting-cell (APC)-based cancer vaccines. Considering the limited scope of immunization using defined peptides, we have studied an alternative approach of specific immunization with tumor-lysate-loaded autologous APC (adherent peripheral mononuclear cells cultured in 1000 U granulocyte/macrophage-colony-stimulating factor for 14 days) as a surrogate vaccine. Seventeen patients (11 with active metastatic disease) were intradermally immunized with the vaccine in a phased dose escalation (10⁵–10⁷ cells/injection) monthly for 4 months. Thirteen patients completed all four immunizations showing no toxicity (3 patients had to be taken off study because of progressive disease and 1 patient went off study as a result of myocardial infarction due to multi-vessel coronary artery disease). None has shown any immediate or delayed toxicity attributable to the immunization and none has shown any evidence of autoimmunity. One patient showed a partial regression of a subcutaneous nodule. Thirteen patients are alive after 4+ months to 30+ months (17-month median survival for the group). Nine patients showed evidence of delayed-type hypersensitivity at the vaccine sites. Monitoring of biological response in conventional natural killer or cytolytic T lymphocyte assays with pre- and post-immune peripheral blood lymphocytes revealed no consistent differences. The vaccine-infiltrating lymphocytes (VIL) from nine specimens were adequately expanded following in vitro stimulation with the respective autologous-lysate-loaded APC for phenotypic and functional analyses. Five of the nine ex vivo expanded VIL were predominantly CD8⁺. Evidence of an antigen-specific CD8⁺ T cell response (cytotoxicity and/or tumor necrosis factor production) was detected in three of the five CD8⁺ VIL. These observations suggest that this type of vaccine is feasible, that it has biological activity, and that the approach may be improved through additional strategic manipulations. Received: 27 March 1998 / Accepted: 14 May 1998     

The type 3 secretion effector IpgD promotes S. flexneri dissemination

The bacterial pathogen Shigella flexneri causes 270 million cases of bacillary dysentery worldwide every year, resulting in more than 200,000 deaths. S. flexneri pathogenic properties rely on its ability to invade epithelial cells and spread from cell to cell within the colonic epithelium. This dissemination process relies on actin-based motility in the cytosol of infected cells and formation of membrane protrusions that project into adjacent cells and resolve into double-membrane vacuoles (DMVs) from which the pathogen escapes, thereby achieving cell-to-cell spread. S. flexneri dissemination is facilitated by the type 3 secretion system (T3SS) through poorly understood mechanisms. Here, we show that the T3SS effector IpgD facilitates the resolution of membrane protrusions into DMVs during S. flexneri dissemination. The phosphatidylinositol 4-phosphatase activity of IpgD decreases PtdIns(4,5)P₂ levels in membrane protrusions, thereby counteracting de novo cortical actin formation in protrusions, a process that restricts the resolution of protrusions into DMVs. Finally, using an infant rabbit model of shigellosis, we show that IpgD is required for efficient cell-to-cell spread in vivo and contributes to the severity of dysentery.     

Cadaveric study of the arterial anatomy of the upper lip

Arterial distribution of the upper lip was investigated in this study. The location, course, length, and diameter of the superior labial artery and its alar and septal branches were determined on 14 preserved cadaver heads. Another cadaver head was used to show the arterial tree by the colored silicone injection technique. The superior labial artery was the main artery of the upper lip and always originated from the facial artery. The superior labial artery was 45.4 mm in length, with a range from 29 to 85 mm. The mean distance of the origin of the superior labial artery from the labial commissura was 12.1 mm. The superior labial artery was 1.3 mm in external diameter at its origin. The mean distance of origin of the superior labial artery from the lower border of the mandible was 46.4 mm. The alar division of the superior labial artery was mostly found as a single branch (82 percent). Its mean length was 14.8 mm and the mean diameter at the origin was 0.5 mm. The distance between the origins of the superior labial artery and the septal branch was 33.3 mm. The septal branch was single in most of the cases (90 percent). The mean length of the septal branch was 18.0 mm and the diameter at its origin was 0.9 mm. After all dissections, it was concluded that the arterial distribution of the upper lip was not constant. The superior labial artery can occur in different locations unilaterally and bilaterally, with the branches showing variability.     

Anatomic study of the vasculature of the submental artery flap

The submental artery island flap is a versatile option in head and neck reconstruction. This flap may be used for the coverage of perioral, intraoral, and other facial defects, leaving a relatively acceptable donor-site scar. In this study, the submental region of 13 formalin-fixed cadavers was dissected bilaterally. Comprehensive anatomical information regarding the pedicle of the flap and its relationship with the important adjacent structures is provided. The mean values of the measurements of the facial and submental arteries were as follows: the facial artery was 2.7 mm in diameter at the origin, and it crossed the mandibular border 26.6 mm from the mandibular angle. The origin of the submental artery was 27.5 mm from the origin of the facial artery, 5.0 mm from the mandibular border, and 23.8 mm from the mandibular angle. The diameter of the submental artery was 1.7 mm at the origin. The artery was found mostly to course superficial to the submandibular gland. In one case, the artery passed through the gland. The total length of the submental artery was 58.9 mm. The artery anastomosed with the contralateral artery in 92 percent of the cadavers. The submental artery was deep to the anterior belly of the digastric muscle in 81 percent of the cases. This study presents detailed anatomical data about the location, dimension, and relationship of the facial artery, the submental artery, and the submental vein that may be useful during dissection of the submental artery island flap.     

The First Isolation of Cryptococcus neoformans from Eucalyptus Trees in South Aegean and Mediterranean Regions of Anatolia in Turkey despite Taurus Mountains alkalinity

Between April 2001 and April 2002 were studied 106 women with a clinical diagnosis of vaginal candidiasis seen at the Gynecology and Obstetrics Ambulatory of the Hospital das Clínicas da Universidade Federal de Goiás. The patients were assessed on two occasions, before starting treatment with itraconazole or fluconazole (initial visit) and 14 days after treatment (return). At two visits the signs and symptoms were recorded and vaginal secretion was collected. According to the clinical evaluation, itraconazole was effective in 64.3%, while fluconazole was effective in 71.0% of the patients. The mycological cure rates (negative culture) in the return were 64.3% for the patients treated with itraconazole and 78.9% for the patients treated with fluconazole. The MICs of itraconazole and fluconazole for 80 Candida isolates were determined by Etest method. We investigated the correlation between in vitro susceptibility (Susceptible, Susceptibility Depending Dose and Resistant) to itraconazole and fluconazole with clinical outcome of the patients. The success rates were 63.9% for itraconazole and 90.6% for fluconazole in the susceptible category, 100.0% for both drugs in the susceptible dose dependent category, and 0.0% for both drugs in the resistant category. Our results showed there were a positive correlation between in vitro susceptibility test results with clinical outcome in vaginal Candida infections and that both drugs might be one choice in the treatment of vaginal candidiasis.     

Reusable nanocopy machine particles for the replication of DNA

As one of the most important components copying DNA molecules in the PCR system, Taq DNA polymerase has a high processivity, however, lower persistence when compared to other polymerases. Studies for the enhancement of stability of Taq DNA polymerase is of great importance. The present study describes the integration of PCR application of cross‐linked Taq DNA polymerase enzyme in a nanochamber using a ruthenium based MATyr‐Ru‐(bipyr)₂)‐MATyr monomer hapten prepared by photosensitive microemulsion polymerization technique. The conjugation and cross‐linking have achieved using our previously invented Aminoacid (monomer) Decorated and Light Underpining Conjugation Approach (ANADOLUCA) method. Microemulsion polymerization media has prepared by dispersing PVA in deionized water. The nano enzyme could be easily prepared at room temperature, in daylight and under nitrogen atmosphere using ruthenium based photosensitive cross‐linking agents. The nano copy machine particles (nano Taq DNA polymerase) are very stable against more acidic or more basic conditions, high temperatures and could be reusable in PCR analysis for many times without any deformation in their structures. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:119–123, 2015     

Serine Carboxypeptidase SCPEP1 and Cathepsin A Play Complementary Roles in Regulation of Vasoconstriction via Inactivation of Endothelin-1

The potent vasoconstrictor peptides, endothelin 1 (ET-1) and angiotensin II control adaptation of blood vessels to fluctuations of blood pressure. Previously we have shown that the circulating level of ET-1 is regulated through its proteolytic cleavage by secreted serine carboxypeptidase, cathepsin A (CathA). However, genetically-modified mouse expressing catalytically inactive CathA S190A mutant retained about 10–15% of the carboxypeptidase activity against ET-1 in its tissues suggesting a presence of parallel/redundant catabolic pathway(s). In the current work we provide direct evidence that the enzyme, which complements CathA action towards ET-1 is a retinoid-inducible lysosomal serine carboxypeptidase 1 (Scpep1), a CathA homolog with previously unknown biological function. We generated a mouse strain devoid of both CathA and Scpep1 activities (DD mice) and found that in response to high-salt diet and systemic injections of ET-1 these animals showed significantly increased blood pressure as compared to wild type mice or those with single deficiencies of CathA or Scpep1. We also found that the reactivity of mesenteric arteries from DD mice towards ET-1 was significantly higher than that for all other groups of mice. The DD mice had a reduced degradation rate of ET-1 in the blood whereas their cultured arterial vascular smooth muscle cells showed increased ET-1-dependent phosphorylation of myosin light chain 2. Together, our results define the biological role of mammalian serine carboxypeptidase Scpep1 and suggest that Scpep1 and CathA together participate in the control of ET-1 regulation of vascular tone and hemodynamics.