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Biomedical subjects

J C Williams

Publications and source records attributed to J C Williams.

At least 19 recordsLinked to original sources
PubMed2001

Long-term transgene expression within the anterior pituitary gland in situ: impact on circulating hormone levels, cellular and antibody-mediated immune responses.

Adenoviral vectors have been identified as useful tools for gene transfer to the pituitary gland with the aim of providing therapeutic treatments for pituitary diseases. Although successful adenovirus-mediated gene transfer to the pituitary has been shown, the duration of transgene expression, local immune responses and consequences on circulating pituitary hormone levels have not been investigated. These are critical not only for the successful implementation of these gene transfer techniques both for physiological and/or therapeutic applications but also for assessing the safety of these approaches. We have therefore assessed duration and levels of transgene expression 3 days, 14 days, 1, 2, and 3 months after delivery of adenoviruses expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK), under the control of the major immediate early human cytomegalovirus (RAd-hCMV/TK) or human PRL (RAd-hPrl/TK) promoters, to the anterior pituitary (AP) gland in situ. The presence of vector genome and cellular immune infiltrates within the AP gland were also studied along with the levels of circulating anti-adenovirus neutralizing antibodies and AP hormones in sera. Ubiquitous or cell-type specific expression of HSV1-TK within the AP gland was seen from RAd-hCMV/TK and RAd-hPrl/TK respectively at all time points, although a reduction in expression was seen over time. PCR amplification of HSV1-TK specific sequences showed the persistence of adenoviral genomes for up to 3 months. Analysis of the AP showed the presence of a virus-induced inflammation that peaked around day 14 and was resolved between 2-3 months. ED1-positive macrophages, CD8-positive T-cells and CD161-positive NK cells were identified up to 1 month after virus administration. A virus-induced humoral immune response was also present as anti-adenovirus neutralizing antibodies were detected from 14 days after virus administration. Levels of circulating pituitary hormones were unaffected by virus administration with the exception of the stress hormone ACTH which was increased at 3 days but normalized by 14 days. In conclusion, our data indicates that adenovirus-mediated delivery to the AP gland in situ may be a useful tool for the treatment of pituitary diseases as no major cytotoxicity or disruption of AP hormonal functions are seen. Despite of this, further developments to this approach still need to be made to combat the reduced transgene expression seen over time and the induction of virus-induced immune responses.

Adenoviridae↗
PubMed2000

Mononuclear cell adhesion to collagen ex vivo is related to pulse pressure in elderly subjects.

Mononuclear cells and platelets are intimately involved in the pathogenesis and complications of cardiovascular disease. Platelet activation has been reported in hypertension, though the activation-state of monocytes has received less attention. In this study the adhesiveness of monocytes and platelets was assessed and any relationship between the adhesive properties of these cellular elements and plasma levels of soluble adhesion molecules and blood pressure parameters determined. Fifty six elderly volunteers, of whom 32 were classified hypertensive (daytime SBP > or = 135 mmHg), underwent 24 h blood pressure monitoring, assessment of monocyte and platelet adhesion and measurement of the plasma soluble adhesion molecules ICAM-1, L-selectin, E-selectin and vWF. In the elderly hypertensive subjects, monocyte adhesion to collagen coated (P < 0.05) and tissue culture plastic microwells (P < 0.05) was significantly elevated and circulating levels of soluble ICAM-1 (P < 0.01) and soluble E-selectin (P < 0.05) were significantly raised compared to their normotensive counterparts. A significant correlation was found to exist between monocyte adhesion to collagen and daytime pulse pressure (r = 0.39, P < 0.01) and also between plasma levels of soluble E-selectin and clinic DBP (r = 0.40, P < 0.001). The increased monocyte adhesion witnessed in hypertensive subjects and with increasing pulse pressure may contribute to the increased risk of cardiovascular disease in hypertension. Whether this increased adhesiveness is a property of the monocytes. or reflects endothelial cell activation, remains to be determined.

Aged↗
PubMed2000

Calcium stone fragility is predicted by helical CT attenuation values.

BACKGROUND AND PURPOSE: Helical CT has become the preferred method for imaging urinary calculi, and so it would be useful if data from helical CT could also be used to predict the number of shockwaves (SWs) needed to break a given stone. METHODS AND MATERIALS: We measured the number of SWs required to comminute calcium stones in vitro. RESULTS: The SW requirement correlated with stone size (volume, weight, diameter) and with helical CT attenuation values when the scans were performed at 3-mm collimation. When CT scans were performed at 1-mm collimation, the number of SWs needed for comminution did not correlate with helical CT attenuation values. This result indicates that the correlation with 3-mm scans was attributable to volume-averaging effects, in which smaller stones yield smaller attenuation values. That is, attenuation values from helical CT at larger beam collimation widths contain information about stone size that can be exploited to predict the fragility of calcium stones. We observed that for calcium stones, the number of SWs to comminution was generally less than half the stone CT attenuation value in Hounsfield units. This "half-attenuation rule" predicted the number of SWs needed to complete fragmentation for 95% of calcium stones (24/24 calcium oxalate monohydrate, 13/13 hydroxyapatite, 8/10 brushite stones). CONCLUSION: This in vitro study suggests that it may be possible to predict effective SW dose using helical CT prior to lithotripsy.

Humans↗
PubMed2000

Effect of vitamin C on ambulatory blood pressure and plasma lipids in older persons.

OBJECTIVES: To determine the effect of oral vitamin C supplements on ambulatory blood pressure and plasma lipids. DESIGN: A 6-month double-blind randomized placebo-controlled cross-over study with a 1 -week washout between cross-over periods. METHODS: Vitamin C 500 mg daily or matching placebo was given to 40 men and women aged between 60 and 80 years for 3 months each in a cross-over fashion. Clinic and 24-h ambulatory blood pressure, plasma ascorbate and lipids were measured at baseline and at the end of each cross-over phase. RESULTS: Clinic blood pressure did not change between placebo and vitamin C phases. Daytime ambulatory blood pressure showed a small but significant fall in systolic blood pressure (2.0 +/- 5.2 mmHg; 95% confidence interval 0-3.9 mmHg) but not in diastolic blood pressure. Regression analysis showed that with increasing baseline daytime blood pressure the fall in blood pressure with vitamin C supplementation increased. Regression analysis of the change in high-density lipoprotein (HDL) cholesterol showed a significant effect of sex on the change in HDL cholesterol. In women, but not men, HDL cholesterol increased significantly by 0.08 +/- 0.11 mmol/l, P=0.007. There was no change in low-density lipoprotein cholesterol between treatment periods. CONCLUSION: In older adults high intakes of ascorbic acid have modest effects on lowering high systolic blood pressure, which could contribute to the reported association between higher vitamin C intake and lower risk of cardiovascular disease and stroke.

Aged↗
PubMed2000

Linkage of an alphavirus host-range restriction to the carbohydrate-processing phenotypes of the host cell.

The Sindbis virus mutant NE2G216 retains PE2 in place of E2 in its virion structure. NE2G216 is a host-range mutant that replicates with near-normal kinetics in vertebrate cells, but displays severely restricted growth in cultured mosquito cells (C6/36) due to defects in the virus maturation process. In this study we tested the hypothesis that the host-range phenotype of NE2G216 was linked to the differences in carbohydrate-processing phenotypes between vertebrate and arthropod cells. Arthropod cell-derived glycoproteins are distinguishable from those synthesized in vertebrate cells by the absence of complex- and hybrid-type N-linked oligosaccharides. To test our hypothesis we compared the growth of the wild-type virus, TRSB, NE2G216 and three PE2-containing, C6/36 cell-adapted variants, in vertebrate cells treated with 1-deoxymannojirimycin (1-dMM). 1-dMM inhibits the Golgi alpha-mannosidase I enzyme and limits oligosaccharide processing to high-mannose forms (Man(8-9)GlcNAc(2)). The growth of TRSB was not restricted by the action of 1-dMM; however, NE2G216 was restricted in a dose-dependent manner. In contrast, the growth of each PE2-containing, C6/36 cell-adapted mutant was enhanced by low concentrations of 1-dMM (up to 1500%) and was only slightly affected by the higher concentrations. These results demonstrate that virion maturation functions of NE2G216 are sensitive to the structure of cis-linked oligosaccharides, and indicate that the carbohydrate-processing phenotypes of the host cell can influence viral host-range and function as a selective pressure in alphavirus evolution.

1-Deoxynojirimycin↗
PubMed2000

Expression of the two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant Venezuelan equine encephalitis virus replicon particles.

RNA replicon particles derived from a vaccine strain of Venezuelan equine encephalitis virus (VEE) were used as a vector for expression of the major envelope proteins (G(L) and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G(L)/M). Open reading frame 5 (ORF5) encodes the G(L) protein, which expresses the known neutralizing determinants of EAV (U. B. R. Balasuriya, J. F. Patton, P. V. Rossitto, P. J. Timoney, W. H. McCollum, and N. J. MacLachlan, Virology 232:114-128, 1997). ORF5 and ORF6 (which encodes the M protein) of EAV were cloned into two different VEE replicon vectors that contained either one or two 26S subgenomic mRNA promoters. These replicon RNAs were packaged into VEE replicon particles by VEE capsid protein and glycoproteins supplied in trans in cells that were coelectroporated with replicon and helper RNAs. The immunogenicity of individual replicon particle preparations (pVR21-G(L), pVR21-M, and pVR100-G(L)/M) in BALB/c mice was determined. All mice developed antibodies against the recombinant proteins with which they were immunized, but only the mice inoculated with replicon particles expressing the G(L)/M heterodimer developed antibodies that neutralize EAV. The data further confirmed that authentic posttranslational modification and conformational maturation of the recombinant G(L) protein occur only in the presence of the M protein and that this interaction is necessary for induction of neutralizing antibodies.

Animals↗
PubMed2000

Effect of vitamin E supplementation on circulating cell adhesion molecules pre- and post-coronary angioplasty.

The soluble adhesion molecules P-selectin (sP-selectin) and intercellular adhesion molecule-1 (sICAM-1) are derived from platelets and endothelial cells. Circulating concentrations of these soluble adhesion molecules are raised in patients with atherosclerosis and following percutaneous transluminal coronary angioplasty (PTCA). We have investigated the effects of vitamin E supplements (800 IU/day) on circulating plasma ICAM-1 and P-selectin levels pre- and post-PTCA. Patients, randomized to group, were pre-treated with vitamin E or placebo (soybean oil) for 1 month before routine PTCA. Plasma sICAM-1 and sP-selectin were measured by enzyme-linked immunosorbent assay on blood taken immediately pre- and post-PTCA. Total protein and alpha-tocopherol were measured on the same samples. Plasma alpha-tocopherol concentrations increased in patients receiving vitamin E: 19.1 (1.5) [mean (standard error of the mean, SEM)] mg/mL post-PTCA versus 13.9 (0.6) mg/mL pre-PTCA (n=23; P<0.01). Plasma sP-selectin and sICAM-1 levels were not significantly increased following PTCA in the vitamin E group. Pre-angioplasty mean (SEM) plasma sP-selectin concentration in the vitamin E group was 8.83 (0.97) ng/mg protein; the corresponding mean post-angioplasty value was 9.34 (0.89) ng/mg protein (P=0.85). The mean (SEM) pre-angioplasty sICAM-1 concentration in this group was 2.18 (0.24) ng/mg protein, and was 2.20 (0.23) ng/mg protein following angioplasty (P = 0.84). In the placebo group (n = 24) there was a significant increase in mean (SEM) sP-selectin concentration following angioplasty, from 7.48 (0.73) to 9.70 (0.78) ng/mg protein (P<0.05). The change (mean, SEM) in plasma sP-selectin concentration following angioplasty was significantly greater for the placebo group [2.22 (0.50) ng/mg protein] than for the group receiving vitamin E [0.50 (0.50) ng/mg protein] (P<0.02). This difference remained significant (P<0.05) even after adjustment for pre-angioplasty P-selectin concentrations. Mean (SEM) plasma sICAM-1 concentrations remained unchanged following angioplasty [pre-angioplasty: 2.16 (0.20) ng/mg protein; post-angioplasty: 1.97 (0.13) ng/mg protein]. Vitamin E may therefore limit platelet or endothelial activation during PTCA.

Adult↗
PubMed2000

Helical CT of urinary calculi: effect of stone composition, stone size, and scan collimation.

OBJECTIVE: Helical CT has become the preferred methodology for identifying urinary calculi. However, the ability to predict stone composition, which influences patient treatment, depends on the accurate measurement of the radiographic attenuation of stones. We studied the effects of stone composition, stone size, and scan collimation width on the measurement of attenuation in vitro. MATERIALS AND METHODS: One hundred twenty-seven human urinary calculi of known composition and size were scanned at 120 kVp, 240 mA, and a 1:1 pitch at different collimations. A model, based on the physics of helical CT, was used to predict the effect of scan collimation width and stone size on measured attenuation. RESULTS: At a 1-mm collimation, stone groups could be differentiated by attenuation: the attenuation of uric acid was less than that of cystine or struvite, which overlapped; these were less than the attenuation of calcium oxalate monohydrate, which was in turn lower than that of brushite and hydroxyapatite, which overlapped and showed the highest values. At a wider collimation, attenuation was lower and the ability to differentiate stone composition was lost. Attenuation also decreased with smaller stones. At a 10-mm collimation, some uric acid stones (<approximately 6 mm) and other stones (< approximately 4 mm) had very low attenuation, so low that they could remain undetected on helical CT. The model predicted well the degree that attenuation was affected by stone size and collimation width. CONCLUSION: Stone composition and stone size, relative to CT collimation, independently influenced CT attenuation. The effect of stone size and collimation generally conformed to the model's predictions. We determined that small stones with low attenuation can be overlooked on helical CT.

Humans↗
PubMed1999

A comparison of persistent anthelmintic efficacy of topical formulations of doramectin, ivermectin, eprinomectin and moxidectin against naturally acquired nematode infections of beef calves.

Persistent anthelmintic efficacy of topical formulations (all at a dosage of 500 microg/kg) of doramectin (DOR), ivermectin (IVM), eprinomectin (EPR) and moxidectin (MOX), in comparison with untreated control cattle (CONT), was observed in stocker beef calves during a 112-day winter-spring grazing trial. Five groups of 15 calves per group were grazed on 15 separate 2 ha pastures following random assignment of animals to specific pastures and then to treatment groups. All of the 5 treatments were represented in each of the 15 pastures. All cattle were weighed on study Days 1, 0, 28, 56, 84, 111 and 112. Fecal samples for nematode egg counts were collected on Days 7, 0, at 7 day intervals through Day 56 and at 14 day intervals to Day 1 12. Pooled group fecal cultures for determining generic composition of nematode infections were prepared at 14 day intervals throughout the study. As based on fecal egg counts, anthelmintic activity of EPR and MOX was greater (p < 0.05) than values for IVM or CONT through Day 28. Activity of DOR was greater (p < 0.05) than that of IVM on Days 7 and 14 only. Although significance levels varied little among treated groups from Day 42 to the end of the study, egg counts and percent reduction values of EPR and MOX remained consistently lower numerically than egg counts and higher than reduction values respectively, of DOR and IVM through Day 70. From Day 70 on, IVM counts were numerically, but not significantly higher than values of CONT. Based on larval culture, Cooperia predominated from Day 0 through 28 and again from Days 70 to 98; Ostertagia was second in prevalence with highest percentages, which exceeded those of Cooperia, between Days 42 and 70. Bodyweights of all treated groups, with exception of IVM, were always significantly greater (p < 0.05) than weights of CONT. Weights of IVM were numerically greater, but not significantly greater than CONT only on Days 84 and 112. From Day 56 on, there were no significant differences between weights of DOR, EPR and MOX, however, numerical values for MOX were consistently higher than values for the other two. Final average total bodyweight gains were: 153.7 kg for MOX, 148.5 kg for EPR, 146.9 kg for DOR, 139.7 kg for IVM and 127.7 kg for CONT.

Administration, Topical↗
PubMed1999

P+HA- charge recombination reaction rate constant in Rhodobacter sphaeroides reaction centers is independent of the P/P+ midpoint potential.

The kinetics of the P+HA- (oxidized donor, reduced bacteriopheophytin acceptor) recombination reaction was measured in a series of reaction center mutants of Rhodobacter sphaeroides with altered P/P+ midpoint potentials between 410 and 765 mV. The time constant for P+HA- recombination was found to range between 14 and 26 ns and was essentially independent of P/P+ midpoint potential. Previous work has shown that the time constant for initial electron transfer in these mutants at room temperature is also only weakly dependent on the P/P+ midpoint potential, ranging from about 2.5 ps to about 50 ps. These results, taken together, imply that heterogeneity in the P/P+ midpoint potential within the reaction center population is not likely the dominant cause of the substantial kinetic complexity observed in the decay of the excited singlet state of P on the picosecond to nanosecond time scale. In addition, the pathway of P+HA- decay appears to be direct or via P+BA- rather than proceeding back through P, even in the highest-potential mutant, as is evident from the fact that the rate of P+HA- recombination is unaltered by pushing P+HA- much closer to P in energy. Finally, the midpoint potential independence of the P+HA- recombination rate constant suggests that the slow rate of P+HA- recombination arises from an inherent limitation in the maximum rate of this process rather than because it occurs in the inverted region of a classical Marcus rate vs free energy curve.

Electron Transport↗
PubMed1999

Effect of macroscopic air bubbles on cell lysis by shock wave lithotripsy in vitro.

In studies of cells or stones in vitro, the material to be exposed to shock waves (SWs) is commonly contained in plastic vials. It is difficult to remove all air bubbles from such vials. Because SWs reflect at an air-fluid interface, and because existing gas bubbles can serve as nuclei for cavitation events, we sought to determine in our system whether the inclusion of small, visible bubbles in the specimen vial has an effect on SW-induced cell lysis. We found that even small bubbles led to increased lysis of red blood cells (1- to 3-mm diameter bubbles, 9.8+/-0.5% lysis, n = 7; no bubbles, 4.4+/-0.8%, n = 4), and that the degree of lysis increased with bubble size. Damage could not be reduced by centrifuging the cells to the opposite end of the vial, away from the bubble. B-scan ultrasound imaging of blood in polypropylene pipette bulbs showed that, with each SW, bubbles were recruited from the air interface, mixing throughout the fluid volume, and these appeared to serve as nuclei for increased echogenicity during impact by subsequent SWs; thus, bubble effects in vials could involve the proliferation of cavitation nuclei from existing bubbles. Whereas injury to red blood cells was greatly increased by the presence of bubbles in vials, lytic injury to cultured epithelial cells (LLC-PK1, which have a more complex cytoarchitecture than red blood cells) was not increased by the presence of small air bubbles. This suggests different susceptibility to SW damage for different types of cells. Thus, the presence of even a small air bubble can increase SW-induced cell damage, perhaps by increasing the number of cavitation nuclei throughout the vial, but this effect is variable with cell type.

Epithelial Cells↗
PubMed1999

Cell damage by lithotripter shock waves at high pressure to preclude cavitation.

Acoustic cavitation has been implicated as a cause of cell damage by lithotripter shock waves, particularly under in vitro conditions. When red blood cells were exposed to shock waves (from an electrohydraulic lithotripter) while under high hydrostatic pressure (> 80 atm), cell lysis was dramatically reduced over that seen at atmospheric pressure, which is consistent with damage due to acoustic cavitation. However, even at > 120 atm of pressure, lysis was still 97% above that of cells not exposed to shock waves, revealing significant damage that apparently was due to mechanisms other than cavitation. Hydrostatic pressure alone did not cause cell lysis, and shock-wave-dependent damage occurred when the cells were in fluid suspension, or when they were centrifuged to the end of the vial. Shock-wave damage at high pressure increased with increasing shock-wave number, and was seen at 24 and 20 kV, but not at 16 kV. This shock-wave damage at high pressure makes up a noteworthy portion of the total cell injury seen at atmospheric pressure (about 10% at 24 kV), suggesting significant noncavitational injury to cells in vitro. Because cavitation occurs far more readily in vitro than in vivo, the noncavitational damage seen in the present study could represent a substantial portion of cell injury seen in vivo with shock-wave lithotripsy.

Erythrocytes↗
PubMed1999

Long-term neural recording characteristics of wire microelectrode arrays implanted in cerebral cortex.

This paper describes a detailed protocol for obtaining chronic, multi-site unit recordings in cerebral cortex of awake animals for periods of three months or more. The protocol includes details for making relatively simple and inexpensive implantable multichannel electrodes that consist of arrays of separate microwires. The results reported in this paper suggest that a viable implant will have discriminable unit activity on about 80% of the electrodes, resulting in, on average, the simultaneous unit recording of upwards of 60 units during a daily recording session. The active electrodes during one recording session tend to remain active in subsequent recording sessions for several weeks. Using the methods described here, implants have been constructed which incorporate several different electrode materials, coatings, sizes, and electrode separation within a single array. These microwire electrode arrays provide the basic technology for obtaining unit recordings for several months. This provides a model system for studying biocompatibility of neural implants, which is a critical component for the development of neural implants that have an indefinite working span.

Action Potentials↗
PubMed1999

Effects of vitamin E on human platelet and mononuclear cell responses in vitro.

Recent epidemiological studies have provided evidence supporting the potential benefits of antioxidants in coronary prevention. We have investigated the effects of vitamin E on platelets, monocytes and endothelial cells in vitro. Pre-incubation of platelets with vitamin E inhibited subsequent thrombin- (P < 0.05, n = 5), collagen- (P < 0. 0001, n = 5) and ADP-(P < 0.05, n = 4) induced platelet aggregation measured using a microtitre plate method, or conventional aggregometry. The adhesion of thrombin-activated platelets to collagen was also inhibited by vitamin E (P < 0.05, n = 8), but not by vitamin C (P > 0.05, n = 8); nor was the adhesion of unstimulated platelets significantly affected (P > 0.05, n = 8). Pre-incubation of monocytes with vitamin E inhibited their subsequent adhesion to plastic (P < 0.05, n = 9), and was also associated with an 18% reduction in adhesion to EA.hy 926 endothelial cells (n = 8), although this failed to reach statistical significance. Pre-incubation of the endothelial cells with vitamin E also significantly reduced subsequent mononuclear cell adhesion by 56% (P < 0.05, n = 3).

Adult↗
PubMed1999

Structural and mutagenesis studies of leishmania triosephosphate isomerase: a point mutation can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power.

The dimeric enzyme triosephosphate isomerase (TIM) has a very tight and rigid dimer interface. At this interface a critical hydrogen bond is formed between the main chain oxygen atom of the catalytic residue Lys13 and the completely buried side chain of Gln65 (of the same subunit). The sequence of Leishmania mexicana TIM, closely related to Trypanosoma brucei TIM (68% sequence identity), shows that this highly conserved glutamine has been replaced by a glutamate. Therefore, the 1.8 A crystal structure of leishmania TIM (at pH 5.9) was determined. The comparison with the structure of trypanosomal TIM shows no rearrangements in the vicinity of Glu65, suggesting that its side chain is protonated and is hydrogen bonded to the main chain oxygen of Lys13. Ionization of this glutamic acid side chain causes a pH-dependent decrease in the thermal stability of leishmania TIM. The presence of this glutamate, also in its protonated state, disrupts to some extent the conserved hydrogen bond network, as seen in all other TIMs. Restoration of the hydrogen bonding network by its mutation to glutamine in the E65Q variant of leishmania TIM results in much higher stability; for example, at pH 7, the apparent melting temperature increases by 26 degrees C (57 degrees C for leishmania TIM to 83 degrees C for the E65Q variant). This mutation does not affect the kinetic properties, showing that even point mutations can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power at the mesophilic temperature.

Animals↗
PubMed1999

Mitochondrial disease and cyclic vomiting syndrome.

Mutations of mitochondrial DNA are being increasingly recognized as a cause of human disease. Six unrelated children have been evaluated with cyclic vomiting syndrome and a strong maternal family history suggesting a mitochondrial DNA mutation. Manifestations suggestive of migraine were present in each child. Additional clinical findings present in all cases include: developmental delay (3/6 cases), seizures (3/6), and poor growth (3/6). The age of onset for vomiting episodes was < or = 1 year in five cases. An elevated body fluid lactate (lactic acid) was found in 5/6 cases. A mitochondrial DNA mutation was confirmed in one child with the finding of a large rearrangement. These cases suggest that mitochondrial DNA mutations can cause cyclic vomiting syndrome. Mitochondrial disease should be considered in cases of cyclic vomiting, especially those with additional pathology or possible maternal inheritance. Initial screening should include plasma lactate and urine organic acids obtained during an episode.

Adolescent↗