General principles in the characterization and use of model systems for biopharmaceutical studies.
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Animals↗
Biomedical subjects
G Wilson
Publications and source records attributed to G Wilson.
Time-domain reflectometry studies on Halobacterium halobium and Halobacterium marismortui.
The dielectric properties of Halobacterium halobium and Halobacterium marismortui measured over the frequency range 1 MHz to 1 GHz are compared with a single-shell model for interfacial polarization. In the case of Halobacterium halobium, the model shows excellent agreement with the experimental data for reasonable values of membrane and cytoplasmic conductivity and permittivity. For Halobacterium marismortui however, an acceptable fit to the data can only be achieved by invoking a plasma membrane conductivity some three orders of magnitude larger than that of Halobacterium halobium and adding a second high-frequency dielectric dispersion. These observations confirm the findings of a previous study and are consistent with a scheme for Halobacterium marismortui involving thermodynamic compartmentalization of Na and K ions and the existence of a conductive plasma membrane.
Cell Membrane↗
Over-the-counter availability of antibiotics.
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Anti-Bacterial Agents↗
The effect of osmolality on the shedding rate of the corneal epithelium.
It has been stated that in dry eye hyperosmolality damages the epithelium of the ocular surface by increasing the rate at which cells are shed. To test this hypothesis, paired excised rabbit corneas were superfused with balanced salt solutions in which all conditions were held constant except the osmolality in contact with the epithelium. For a period of 400 min the epithelium of one of the corneas was exposed to one of seven test osmolalities (200-425 mOsm/kg), whereas the other cornea was used as a control (305 mOsm/kg). The number of cells shed from each corneal surface was counted, and thickness changes in the epithelium and stroma were measured. Only hypoosmotic solutions of 260 mOsm/kg or less increased the number of cells shed relative to the control. None of the hyperosmotic solutions significantly increased the shedding rate. There was no significant change in the thickness of the epithelium, whereas the stroma swelled in hypoosmotic solutions and thinned in hyperosmotic solutions. It is concluded that hyperosmolality in the range encountered in dry eyes is not sufficient in itself to increase the shedding rate.
Animals↗
Anti-GM1 ganglioside antibodies cloned from autoimmune neuropathy patients show diverse binding patterns in the rodent nervous system.
We have recently cloned a panel os monoclonal IgM anti-GM1 ganglioside antibodies from peripheral blood lymphocytes of patients with multifocal motor neuropathy and Guillain Barré syndrome. In solid-phase immunoassay, the antibodies all reacted with GM1 and also reacted to different degrees with the structurally related glycolipids asialo-GM1 and GD1b. These antibodies are being used to study the pathogenesis of the anti-GM1 antibody-medicated neuropathy in different experimental systems. In the present immunofluorescence study we report the binding patterns of 5 of these antibodies in the rodent nervous system. The antibodies demonstrated highly diverse binding patterns on tissue sections and teased fibers when compared to one another and between species. The antibodies bound many central and peripheral nervous system structures, including neurons and myelin, motor end plate regions, and muscle spindles. The diversity of binding shown by these antibodies provide evidence that may account for the differing clinical phenotypes, including normality, associated with elevated titers of anti-GM1 antibodies.
Animals↗
Mandibular invasion diagnosed by SPECT.
To date no investigation has proven accurate and reliable for assessing mandibular invasion by carcinoma prior to surgery. This prospective study compared a new imaging modality, single photon emission computed tomography (SPECT), with clinical examination and high resolution CT scanning to evaluate the sensitivity and specificity of each method in detecting mandibular invasion by squamous cell carcinoma. Twenty-nine patients (21 men, 8 women) with an oral cavity/oropharyngeal carcinoma were studied. All underwent primary surgery and/or radiotherapy where indicated. Resected mandibles were decalcified and examined for tumour infiltration. Imaging studies were read independently by two experienced observers. Clinical examination predicted bone invasion with a sensitivity of 90% and a specificity of only 25%. CT had a sensitivity of 89% and a specificity of 57% while SPECT imaging had a sensitivity of 100% and a specificity of 29%. Using Fisher's exact test and the Kappa statistic for pairwise comparisons between each method, SPECT imaging was complementary to CT in the pre-operative assessment of mandibular invasion.
Aged↗
Chemical approaches to improve the oral bioavailability of peptidergic molecules.
This review discusses both tools and strategies that may be employed as approaches towards the pursuit of orally active compounds from peptidergic molecules. Besides providing a review of these subjects, this paper provides an example of how these were utilized in a research programme at SmithKline Beecham involving the development of orally active GPIIb/IIIa antagonists. The tools for studying oral drug absorption in-vitro include variants of the Ussing chamber which utilize either intestinal tissues or cultured epithelial cells that permit the measurement of intestinal permeability. Example absorption studies that are described are mannitol, cephalexin, the growth hormone-releasing peptide SK&F 110679 and two GPIIb/IIIa antagonist peptides SK&F 106760 and SK&F 107260. With the exception of cephalexin, these compounds cross the intestine by passive paracellular diffusion. Cephalexin, on the other hand, crosses the intestine via the oligopeptide transporter. Structure-transport studies are reviewed for this transporter. The tools for studying oral drug absorption in-vivo involve animals bearing in-dwelling intestinal or portal vein catheters. A study of the segmental absorption of SK&F 106760 is provided. The review concludes with two chemical strategies that may be taken towards the enhancement of oral bioavailability of peptidergic molecules. The first strategy involves the chemical modification of peptides which enhance intestinal permeability, specifically the modification of amide bonds. The second strategy involves the design of compounds bearing nonpeptide templates, which are more amenable to the discovery of compounds with oral activity, from peptide pharmacophore models. An example is given regarding the discovery of SB 208651, a potent orally active GPIIb/IIIa antagonist, designed from the peptides SK&F 106760 and SK&F 107260.
Absorption↗
Incidence of persistent symptoms after laparoscopic cholecystectomy: a prospective study.
BACKGROUND AND AIMS: Laparoscopic cholecystectomy is the standard treatment for symptomatic gall stone disease. This study aimed to assess the effect of the operation on patients' symptoms. METHODS: One hundred consecutive patients undergoing laparoscopic cholecystectomy between June 1994 and June 1995 were evaluated using standard questionnaires examining demographic details, indication for laparoscopic cholecystectomy, characteristics of pain, and other associated dyspeptic and colonic symptoms. A history of psychiatric disturbances and of hysterectomy were also recorded. The same questionnaires were administered again six months after the operation. Operation notes and histological reports were reviewed. RESULTS: Three patients were converted to open surgery and were excluded from analysis. The median age of the remaining 97 patients was 50.9 (19-85) years; 19 were men. There was one complication each of bleeding and biliary leak. Indications for laparoscopic cholecystectomy were biliary type pain (66 patients) and complicated gall stone disease (acute cholecystitis 21, cholestatic jaundice six, and pancreatitis four). Thirteen patients (13%) had persistent pain and two (3%) developed diarrhoea at follow up. Only one patient with persistent pain after laparoscopic cholecystectomy originated from the complicated gall stone disease group. Logistic discriminant analysis showed that bloating (p < 0.001), constipation (p < 0.05), and previous and current use of psychotrophic drugs (p < 0.001) were significantly more common among those with a poor outcome after laparoscopic cholecystectomy. Heartburn was unaffected. Of patients with persistent symptoms after cholecystectomy 77% had no or mild histological changes of cholecystitis as compared with 30% in the pain free group. CONCLUSIONS: The incidence of persistent pain after laparoscopic cholecystectomy was 13%. Abdominal bloating and psychiatric medications were predictive for persistence of pain after laparoscopic cholecystectomy.
Adult↗
Relationship among hormonal treatments, suppression of spermatogenesis, and testicular protection from chemotherapy-induced damage.
To further elucidate the mechanism by which hormonal pretreatment protects the rat testis from damage by procarbazine, we investigated the relationship between the suppression of hormone levels and spermatogenesis and the recovery of spermatogenesis from stem spermatogonia. LBNF1 rats were implanted with capsules containing testosterone or testosterone plus estradiol. After hormone treatment, rats were injected with procarbazine, and recovery of spermatogenesis was assessed. Testosterone (2 cm) plus estradiol (0.5-cm) reduced serum LH levels causing intratesticular testosterone (ITT) to fall to 3% of control levels within 2 weeks, but testis weights and sperm head counts were not appreciably suppressed until 4 weeks. Two weeks' hormone pretreatment, only slightly enhanced spermatogenesis recovery, but 4 weeks markedly increased it. Testosterone (2 cm) alone produced slower suppression of spermatogenesis and less protection from procarbazine than did testosterone plus estradiol implants, despite equivalent suppression of LH and ITT. Long testosterone implants (24-cm) partially maintained ITT at 14% of control despite undetectable LH levels, prevented any decline in sperm counts, and nearly completely abrogated the protective effect of the hormone treatment. Protection appeared to be best correlated with the testis weight reduction by hormone treatment. Thus, recovery of spermatogenesis after chemotherapy is dependent on the degree of suppression of spermatogenesis caused by the reduction of ITT levels at the time of chemotherapy and likely involves cells, such as the Sertoli cells, that are both androgen-responsive and affected by the numbers of germ cells present.
Animals↗
The cell shedding rate of the corneal epithelium--a comparison of collection methods.
PURPOSE: To determine the spontaneous shedding rate of cells from the rabbit corneal epithelium using different harvesting methods. METHODS: Cells were collected from the rabbit corneal epithelium using two in vitro methods and three in vivo methods. Cells were counted and the shedding rate calculated after adjustment for the collection time. RESULTS: The shedding rates obtained from the in vitro methods were (cells/min/cornea, mean +/- SE): 78.0 +/- 9.4 (corneal superfusion), and 5.4 +/- 1.3 (whole-eye perfusion). For the in vivo corneas, the shedding rates were: 10.0 +/- 2.3 (corneal superfusion), 8.1 +/- 0.4 (corneal immersion), and 14.5 +/- 1.5 (corneal irrigation). In vitro corneal superfusion was significantly different from the other four methods (P < 0.01). CONCLUSIONS: The results suggest that the spontaneous cell shedding rate of the in vivo rabbit corneal epithelium is 5 to 15 cells/min/cornea. This is much lower than estimates of about 100 cells/min/cornea based upon in vitro corneal superfusion. One explanation of this slow shedding rate is that factors which were absent during our collection methods (such as blinking) would normally increase shedding. Another possibility is that cells in the corneal epithelium may have a much longer life span than previously reported; rather than a few days, the epithelium could take several months to completely replace all cells. Whatever the explanation, the measured spontaneous shedding rate does not complement the reported production rate of new cells. It is necessary to revise our understanding of the kinetics of epithelial homeostasis.
Animals↗
Apoptosis in the corneal epithelium.
PURPOSE: To determine the viability of corneal surface cells and to determine whether apoptosis is present in the corneal epithelium of the rabbit. METHODS: The viability of epithelial surface cells was examined using a calcein-ethidium assay. In this two-color fluorescence assay, viable cells fluoresce green, and the nuclei of nonviable cells fluoresce red. The number of nonviable cells on the normal corneal epithelial surface was quantified. Surface and shed cells also were examined from shear-stressed corneas. The TUNEL technique (TdT-mediated dUTP nick-end labeling) was used to detect DNA fragmentation characteristic of apoptotic cells. RESULTS: The calcein-ethidium viability assay revealed that the normal epithelial surface is composed mainly of viable cells, with nonviable cells making up a small percentage of the whole. There was an increase in the density of nonviable cells from the periphery of the epithelial surface to the center, In flatmounts of the cornea, TUNEL labeling demonstrated that a small percentage of surface cells exhibited DNA fragmentation, whereas cross-sections revealed that DNA-fragmented cells were found exclusively on the epithelial surface. Epithelial cells from shear-stressed corneas showed an increased number of apoptotic cells. CONCLUSIONS: The normal epithelial surface consists mainly of viable cells, with only a small percentage of nonviable cells and apoptotic cells. The results suggest that nonviable epithelial cells are shed after terminal differentiation, whereas viable cells can be shed by classical apoptosis with the formation of blebs. Thus, there appears to be more than one mechanism for removal of cells from the corneal surface.
Animals↗
Vibrational Raman optical activity of alpha-lactalbumin: comparison with lysozyme, and evidence for native tertiary folds in molten globule states.
Proteins in aqueous solution are now accessible to Raman optical activity (ROA) measurements, which provide an incisive new probe of secondary and tertiary structure illustrated here by a study of bovine alpha-lactalbumin. The room-temperature ROA spectrum of native bovine alpha-lactalbumin is similar to that of native hen egg-white lysozyme except for features attributable to differences in the loop regions: in particular, a positive ROA band at approximately 1338 cm-1 assigned to conformationally homogeneous loop structure, possibly with local order corresponding to 3(10)-helix, has more than double the intensity in alpha-lactalbumin compared with lysozyme. This is consistent with the two proteins having similar secondary structure but different local details in the tertiary fold. ROA measurements on alpha-lactalbumin at pH 2.0 over a range of temperatures have provided a new perspective on the molten globule state. Thus at 35 degrees C ROA reveals the presence of some secondary structure but an almost complete loss of the tertiary loop structure; whereas at 2 degrees C the ROA spectrum is almost identical with that of the native protein, which is strong evidence that virtually all of the secondary structure and the tertiary backbone fold persist, albeit within a looser framework associated with increased solvent exposure and change of environment of many of the side-chains as evidenced by an increase in noise and bandwidth of some of the ROA signals together with aromatic fluorescence and near-UV circular dichroism signals characteristic of the molten globule state. Our sample of acid alpha-lactalbumin at 2 degrees C therefore appears to be an archetypal example of Ptitsyn's "native-like" molten globule, having a fixed native-like tertiary fold but with loss of tight packing of the side-chains; whereas at 35 degrees C it is a "disordered" molten globule. At 20 degrees C the acid molten globule appears to retain highly native-like secondary structure but with most of the tertiary fold already lost. A calcium-free sample of alpha-lactalbumin at neutral pH displayed a broad cooperative transition between native and molten globule states at approximately 15 degrees C, with the latter state showing similar but somewhat degraded tertiary loop ROA signatures to the native protein. In both the acid and apo molten globule states the ROA signatures of the secondary structure and the tertiary loops showed a gradual change with temperature.
Calcium↗
Analysis of anti-GM1 ganglioside IgM antibodies cloned from motor neuropathy patients demonstrates diverse V region gene usage with extensive somatic mutation.
Serum anti-GM1 ganglioside Abs are abnormally elevated in patients with chronic motor neuropathies and Guillain-Barré syndrome. We have cloned six anti-GM1 IgM Abs from PBMCs of five affected patients. VH and VL gene analysis demonstrated substantial somatic mutation, predominantly clustered around VH CDR2 and FR3 in all six Abs. The Abs SM1, DO1, and WO1, isolated from three different patients, are encoded by closely related VH gene segments with restricted canonical structures. The corresponding L chains are encoded by V lambda II and V lambda VIII genes and also share the same canonical structures, containing nucleotide deletions at the VL/JL boundary to form a VLCDR3 of 10 amino acids. DO1 and WO1 VH segments show nucleotide identities of 95.6% and 88.8%, respectively, to the VH3 member 1.9III, and SM1 shows 87.8% identity to the closely related VH3 member, Hv3005. The Abs BO1 and BO3, derived from a single patient and containing the same VH-D-JH and VL-JL rearrangements, are encoded by the VH3 gene, HHg19, with nucleotide identities of 92.2 and 91.2%, respectively, and have both common and unique somatic mutations. The VH sequence of BR1 has 91.3% identity to the VH4 member, V71-2. These data indicate that neuropathy-associated anti-GM1 IgM Abs can be encoded by both diverse and closely related V genes in association with restricted canonical structures, and that their V genes are somatically mutated, consistent with an origin through an Ag-driven immune response.
Amino Acid Sequence↗
Cell-mediated immunity in combat veterans with post-traumatic stress disorder.
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Humans↗
MRI T staging of squamous cell carcinoma of the oral cavity: radiological-pathological correlation.
Fifty patients with primary or suspected recurrent squamous cell carcinoma of the oral cavity were examined pre-operatively with magnetic resonance imaging (MRI) to determine the tumour stage. Pathological correlation was available in all cases. Twenty-five of 35 patients with primary disease were correctly staged (MR Tstage 0-4). In another four patients there was good size correlation between MR and pathology, but two cases had false positive MR interpretation of bone invasion. MRI has proved accurate at excluding the presence of bone invasion (negative predictive value 97%), but a positive scan may not discriminate between tumour and other dental pathology (positive predictive value 67%). Assessment of local extent was otherwise good with invasion of muscles of the floor of the mouth and muscles of mastication always predicted correctly. Salivary gland abnormality was frequent, and MRI correctly distinguished between obstructive sialectasis and direct invasion. The results of MR staging in patients with recurrent disease were less accurate. Where diagnostic scans can be obtained MRI can provide useful information for the surgeon or radiotherapist regarding the local extent of tumour, and may assist in treatment planning. Assessment of cervical lymphadenopathy can be performed as part of same procedure.
Adult↗
Measuring the fetal kidney with ultrasonography.
OBJECTIVES: To establish a normal range of measurements for the external and internal dimensions of the fetal kidney and, if possible, to correlate these measurements with gestational age. SUBJECTS AND METHODS: The external and internal dimensions of the fetal kidney were measured in 810 women selected on the grounds of obstetric, fetal or technical risk factors from a consecutive series of 1136 women attending the ante-natal ultrasonography clinic of a district general hospital. Post-natal data were obtained from medical records on 1122 newborns. The maximum of pairs of renal measurements was used for analysis. Measurements were cross-sectional in 347 cases and longitudinal in 463. The total number of measurement episodes was 2294. RESULTS: External renal dimension and gestational age were closely related, enabling accurate growth centile charts to be constructed. The correlation between renal pelvic dimension and gestational age was weak. The maximum dimension of the renal pelvis at any gestational age in 92.7% of fetuses was < 5 mm. Using longitudinal measurements, 6.5% of cases with a renal pelvic dimension of < 5 mm at the first scan measured > or = 5 mm at a subsequent scan, but 53.3% with a dimension of > or = 5 mm at the first scan also measured > or = 5 mm at the subsequent scan. CONCLUSION: It is possible to assess the growth and size of the fetal kidney according to gestational age but this does not apply to the renal pelvis. A renal pelvic dimension of > 5 mm at any gestational age is unusual and dilatation beyond this level should prompt a detailed post-natal urological investigation.
Female↗
The use of expandable metal stents for large airway obstruction in older patients.
OBJECTIVE: To review the indications, management, and outcome of 30 older patients who had expandable metal stents inserted for large airway obstruction. DESIGN: Information was collected retrospectively from case notes about presentation, radiographic appearances, pulmonary function, including arterial oxygen tension, and histology. Survival data were collected by reviewing hospital or General Practice records. MEASUREMENTS: Spirometry, peak expiratory flow rate, and blood gases were recorded before and after stent insertion. MAIN RESULTS: There was a significant improvement in the patient's mean forced expiratory volume in 1-second (FEV1) and mean peak expiratory flow rate (PEFR). The arterial oxygen tension (pO2) increased from 8.6 Kpa to 10.6 Kpa. The mean length of survival for the whole group was just under 5 months. CONCLUSIONS: Airway stenting for obstruction provides palliative and functional benefits in these severely disabled patients and a consequent improvement in quality of remaining life.
Aged↗