Characterization of antibodies in single-chain format against the E7 oncoprotein of the Human papillomavirus type 16 and their improvement by mutagenesis

BACKGROUND: Human papillomaviruses (HPV) are the etiological agents of cervical cancer. The viral E7 protein plays a crucial role in viral oncogenesis. Many strategies have been explored to block the E7 oncoprotein activity. The single-chain variable antibody fragments (scFvs) are valuable tools in cancer immunotherapy and can be used as "intracellular antibodies" to knock out specific protein functions. For both in vivo and in vitro employment, the scFv intrinsic solubility and stability are important to achieve long-lasting effects. Here we report the characterization in terms of reactivity, solubility and thermal stability of three anti-HPV16 E7 scFvs. We have also analysed the scFv43 sequence with the aim of improving stability and then activity of the antibody, previously shown to have antiproliferative activity when expressed in HPV16-positive cells. METHODS: The three anti-HPV16 E7 scFv 32, 43 51 were selected from the ETH-2 "phage-display" library. Thermal stability was evaluated with ELISA by determining the residual activity of each purified scFv against the recombinant HPV16 E7, after incubation in the presence of human seroalbumine for different time-intervals at different temperatures. Sequence analysis of the scFvs was performed with BLAST and CLUSTALL programs. The scFv43 aminoacid changes were reverted back to the consensus sequence from the immunoglobuline database by site-directed mutagenesis. ScFv solubility was evaluated with Western blotting by determining their relative amounts in the soluble and insoluble fractions of both prokaryotic and eukaryotic systems. RESULTS: ScFv51 was the most thermally stable scFv considered. Sequence analysis of the most reactive scFv43 has evidenced 2 amino acid changes possibly involved in molecule stability, in the VH and VL CDR3 regions respectively. By mutagenesis, two novel scFv43-derived scFvs were obtained, scFv43 M1 and M2. ScFv43 M2 showed to have improved thermal stability and solubility in comparison with the parental scFv43. CONCLUSION: The characterization of 5 specific anti-HPV16 E7 scFvs shows features important for their activity in vivo. ScFv43 M2 shows higher thermal stability with respect to the parental scFv43, and scFv51 shows high stability and solubility. These properties make the 2 scFvs the best candidates to be tested for anti-E7 activity in vivo

Rh-POP Pincer Xantphos Complexes for C-S and C-H Activation. Implications for Carbothiolation Catalysis

The neutral Rh­(I)–Xantphos complex [Rh­(κ³-_P,O,P-Xantphos)­Cl]_<i>n</i>, <b>4</b>, and cationic Rh­(III) [Rh­(κ³-_P,O,P-Xantphos)­(H)₂]­[BAr^F₄], <b>2a</b>, and [Rh­(κ³-_P,O,P-Xantphos-3,5-C₆H₃(CF₃)₂)­(H)₂]­[BAr^F₄], <b>2b</b>, are described [Ar^F = 3,5-(CF₃)₂C₆H₃; Xantphos = 4,5-bis­(diphenylphosphino)-9,9-dimethylxanthene; Xantphos-3,5-C₆H₃(CF₃)₂ = 9,9-dimethylxanthene-4,5-bis­(bis­(3,5-bis­(trifluoromethyl)­phenyl)­phosphine]. A solid-state structure of <b>2b</b> isolated from C₆H₅Cl solution shows a κ¹-chlorobenzene adduct, [Rh­(κ³-_P,O,P-Xantphos-3,5-C₆H₃(CF₃)₂)­(H)₂(κ¹-ClC₆H₅)]­[BAr^F₄], <b>3</b>. Addition of H₂ to <b>4</b> affords, crystallographically characterized, [Rh­(κ³-_P,O,P-Xantphos)­(H)₂Cl], <b>5</b>. Addition of diphenyl acetylene to <b>2a</b> results in the formation of the C–H activated metallacyclopentadiene [Rh­(κ³-_P,O,P-Xantphos)­(ClCH₂Cl)­(σ,σ-(C₆H₄)­C­(H)CPh)]­[BAr^F₄], <b>7</b>, a rare example of a crystallographically characterized Rh–dichloromethane complex, alongside the Rh­(I) complex <i>mer</i>-[Rh­(κ³-_P,O,P-Xantphos)­(η²-PhCCPh)]­[BAr^F₄], <b>6</b>. Halide abstraction from [Rh­(κ³-_P,O,P-Xantphos)­Cl]_<i>n</i> in the presence of diphenylacetylene affords <b>6</b> as the only product, which in the solid state shows that the alkyne binds perpendicular to the κ³-POP Xantphos ligand plane. This complex acts as a latent source of the [Rh­(κ³-_P,O,P-Xantphos)]⁺ fragment and facilitates <i>ortho</i>-directed C–S activation in a number of 2-arylsulfides to give <i>mer</i>-[Rh­(κ³-_P,O,P-Xantphos)­(σ,κ¹-Ar)­(SMe)]­[BAr^F₄] (Ar = C₆H₄COMe, <b>8</b>; C₆H₄(CO)­OMe, <b>9</b>; C₆H₄NO₂, <b>10</b>; C₆H₄CNCH₂CH₂O, <b>11</b>; C₆H₄C₅H₄N, <b>12</b>). Similar C–S bond cleavage is observed with allyl sulfide, to give <i>fac</i>-[Rh­(κ³-_P,O,P-Xantphos)­(η³-C₃H₅)­(SPh)]­[BAr^F₄], <b>13</b>. These products of C–S activation have been crystallographically characterized. For <b>8</b> in situ monitoring of the reaction by NMR spectroscopy reveals the initial formation of <i>fac</i>-κ³-<b>8</b>, which then proceeds to isomerize to the <i>mer</i>-isomer. With the <i>para</i>-ketone aryl sulfide, 4-SMeC ₆H₄COMe, C–H activation <i>ortho</i> to the ketone occurs to give <i>mer</i>-[Rh­(κ³-_P,O,P-Xantphos)­(σ,κ¹-4-(COMe)­C₆H₃SMe)­(H)]­[BAr^F₄], <b>14</b>. The temporal evolution of carbothiolation catalysis using <i>mer</i>-κ³-<b>8</b>, and phenyl acetylene and 2-(methylthio)­acetophenone substrates shows initial fast catalysis and then a considerably slower evolution of the product. We suggest that the initially formed <i>fac</i>-isomer of the C–S activation product is considerably more active than the <i>mer</i>-isomer (i.e., <i>mer</i>-<b>8</b>), the latter of which is formed rapidly by isomerization, and this accounts for the observed difference in rates. A likely mechanism is proposed based upon these data

Behavioral, physiologic, and stress-related hormonal and metabolic responses to intravenous and epidural morphine in goats

Objective: To establish the effects of epidural and intravenous (IV) morphine on stress-related hormones, metabolites, and behavior in goats. Animals: Six 6-year-old neutered goats (3 males, 3 females) weighing 62.5 +/- 16.5 kg (mean +/- SD). Material and Methods: Study was a prospective cross-over design in which morphine (0.1 mg/kg, epidural and IV) was administered in 2 trials I week apart. Epidural catheters were placed 24 hours before study. Blood samples were collected, physiologic variables were measured, and behavior was scored (0 to 3; 0 = normal, 3 = fractious) at 0, 15, and 30 minutes and 1, 2, 4, 6, and 8 hours (IV trial) and at 0, 1, 2, 4, 6, 8, 12, and 24 hours (epidural trial). Analysis included repeated measures analysis of variance and Tukey-Kramer multiple comparisons test. A P value <0.05 was considered significant. Results: For IV morphine, differences were found for respiratory rate (17 +/- 10 to 27 +/- 11 breaths/min) and temperature. Temperature (38.6 +/- 0.1 degrees C increased at 8 hours (3 9.0 +/- 0.2 degrees C. After epidural morphine, heart rate (96 24 beats/min) decreased at 8 (73 +/- 15 beats/min), 12 (74 +/- 13 beats/min), and 24 (74 +/- 19 beats/min) hours; systolic blood pressure (indirect) (106 +/- 31 mmHg) increased at 6 (157 +/- 18 mmHg), 8 (151 +/- 18 mmHg), and 24 (142 +/- 10 mmHg) hours; diastolic blood pressure (indirect) (71 +/- 23 mmHg) increased at 6 (120 +/- 23 mmHg) and 8 (111 +/- 28 mmHg) hours; and mean blood pressure (indirect) (84 +/- 25 mmHg) increased at 6 (134 +/- 20 mmHg) and 8 (125 +/- 24 mmHg) hours. Glucose concentrations increased from 0 minutes (55.6 +/- 4.6 mg/dL) to 60 minutes (73.1 +/- 15.2 mg/dL) and 120 minutes (70.7 +/- 13.4 mg/dL) before returning to baseline at 720 minutes (55.9 +/- 6.0 mg/dL). Temperature ranged from 38.8 +/- 0.1 degrees C to 39.3 +/- 0.2 degrees C; and cortisol increased from baseline (1.15 +/- 0.91 mu g/dL) to 60 minutes (2.23 +/- 0.78 mu g/dL), which was higher than subsequent cortisol concentrations. Free fatty acids were maximal at 60 minutes (317 +/- 183 mmol/L) and decreased after 480 minutes (145 +/- 28 mmol/L). During the epidural trial, all goats vocalized and changed posture upon injection of the morphine. Conclusions and Clinical Relevance: Behavioral, physiologic, and stress-related hormonal and metabolic responses are minor after IV morphine in goats. Physiologic changes after epidural morphine may interfere with the postoperative evaluation of pain in goats that received epidural morphine. However, it is possible that epidural injection of any substance may initiate the stress-related changes seen in this study

Effect of medetomidine and its antagonism with atipamezole on stress-related hormones, metabolites, physiologic responses, sedation, and mechanical threshold in goats

To evaluate the effects of medetomidine and its antagonism with atipamezole in goats. Prospective randomized crossover study with 1 week between treatments. Six healthy 3-year-old neutered goats (three male and three female) weighing 39.1–90.9 kg (60.0 ± 18 kg, mean ± SD). Goats were given medetomidine (20 μg kg−1, IV) followed, 25 minutes later, by either atipamezole (100 μg kg−1, IV) or saline. Heart and respiratory rate, rectal temperature, indirect blood pressure, and mechanical threshold were measured, and sedation and posture were scored and blood samples obtained to measure epinephrine, norepinephrine, free fatty acids, glucose, and cortisol concentrations at baseline (immediately before medetomidine), 5 and 25 minutes after medetomidine administration, and at 5, 30, 60, and 120 minutes after the administration of antagonist or saline. Parametric and nonparametric tests were used to evaluate data; p < 0.05 was considered significant. Medetomidine decreased body temperature, heart rate, and respiratory rate and increased mean arterial blood pressure, cortisol, and glucose. Recumbency occurred 89 ± 50 seconds after medetomidine administration. All goats were standing 86 ± 24 seconds after atipamezole administration whereas all goats administered saline were sedate and recumbent at 2 hours. Tolerance to compression of the withers and metacarpus increased with medetomidine. From 5 to 120 minutes after saline or atipamezole administration, there were differences in body temperature, glucose, and cortisol but none in heart rate or blood pressure. Three of the six goats receiving saline developed bloat; five of six urinated. After atipamezole, four of six goats developed piloerection and all goats were agitated and vocalized. At the doses used, atipamezole antagonized the effects of medetomidine on recumbency, sedation, mechanical threshold, and the increase in glucose. Atipamezole increased the rate of return of cortisol toward baseline, and prevented further decline in rectal body temperature. Atipamezole may be used to antagonize some, but not all effects of medetomidine

The Effect of Detomidine and its Antagonism With Tolazoline on Stress‐Related Hormones, Metabolites, Physiologic Responses, and Behavior in Awake Ponies

Six ponies were used to investigate the effect of tolazoline antagonism of detomidine on physiological responses, behavior, epinephrine, norepinephrine, Cortisol, glucose, and free fatty acids in awake ponies. Each pony had a catheter inserted into a jugular vein 1 hour before beginning the study. Awake ponies were administered detomidine (0.04 mg/kg intravenously [IV]) followed 20 minutes later by either tolazoline (4.0 mg/kg IV) or saline. Blood samples were drawn from the catheter 5 minutes before detomidine administration (baseline), 5 minutes after detomidine administration, 20 minutes after detomidine administration which was immediately before the administration of tolazoline or saline (time [T] = 0), and at 5, 30, and 60 minutes after injections of tolazoline or saline (T = 5, 30, and 60 minutes, respectively). Compared with heart rate at T = 0, tolazoline antagonism increased heart rate 45% at 5 minutes. There was no difference in heart rate between treatments at 30 minutes. Blood pressure remained stable after tolazoline, while it decreased over time after saline. Compared with concentrations at T = 0, tolazoline antagonism of detomidine in awake ponies resulted in a 55% increase in Cortisol at 30 minutes and a 52% increase in glucose at 5 minutes. The change in free fatty acids was different for tolazoline and saline over time. Free fatty acids decreased after detomidine administration. Free fatty acids did not change after saline administration. After tolazoline administration, free fatty acids increased transiently. Tolazoline tended to decrease sedation and analgesia at 15 and 60 minutes postantagonism. Antagonism of detomidine‐induced physiological and behavioral effects with tolazoline in awake ponies that were not experiencing pain appears to precipitate a stress response as measured by Cortisol, glucose, and free fatty acids. If antagonism of an α‐agonist is contemplated, the potential effect on hormones and metabolites should be considered