Results of intraocular lens implantation in paediatric aphakia. Intraocular lenses were implanted in 16 eyes of 13 patients with congenital cataract, and visual progress was plotted using a preferential-looking technique. Initial surgery was by lens aspiration with preservation of the posterior capsule, and subsequent posterior capsulotomy without anterior vitrectomy. Poly-HEMA posterior chamber lenses were used, usually as a primary procedure but in four cases as a secondary procedure after contact lens failure. No serious complications were encountered. Most eyes achieved a very significant visual improvement, and none were worse than preoperatively. Residual refractive error was highly unpredictable, but did not exceed 6 dioptres. The importance of rigorous occlusion therapy is stressed. With close follow-up, this procedure offers an effective and safe method for the correction of unilateral paediatric aphakia, and, in selected cases only, for bilateral aphakia.


The newer generation intraocular lens power calculation formulae in anterior chamber implants. One hundred and fifty-one eyes with anterior chamber implants and a best corrected visual acuity of at least 6/12 were studied to compare the predictive accuracies of the newer generation intraocular lens power calculation formulae. There was no statistically significant difference between the predictive accuracies of the SRK (Sanders-Retzlaff-Kraff), SRK II, SRK/Theoretical (SRK/T) and Holladay formulae. All these four formulae were more accurate than the Binkhorst II formula.


Peripheral corneal oedema following cataract extraction (Brown-McLean syndrome). We describe the clinical features of 21 eyes of 15 patients who developed peripheral bullous corneal oedema following cataract extraction. The peripheral oedema was asymptomatic in all cases and was only noted at a review examination. Although the onset of the peripheral oedema was probably delayed for several years after surgery, it was not documented to progress in any patient or to involve the central cornea. Endothelial specular microscopy did not reveal any endothelial features that are specific for this condition, but confirmed that the central endothelial cell density can be as high as 2150 cells/mm2 in the presence of peripheral corneal decompensation. Despite the surface irregularity resulting from the peripheral corneal oedema 12 patients (18 eyes) continued to wear contact lenses to correct their aphakia.


Suitability for day case cataract surgery. Areas of concern about day case surgery are highlighted. A group of 442 patients underwent cataract surgery with lens implant. They were randomly allocated to day case or inpatient groups. Questionnaires were used to assess opinions about day case cataract surgery and how patients felt they would manage. None of the areas of concern were actually a problem. Difficulties encountered by patients and clinicians in the postoperative period are discussed. Most patients appear suitable for day case surgery provided they are well informed.


The effect of electromagnetic stimulation of the posterior parietal cortex on eye movements. The posterior parietal cortex probably plays a central role in the sensorimotor transformations needed to make an accurate saccadic eye movement to a visual target. In an attempt to disrupt the normal programming of saccades, we magnetically stimulated the posterior parietal cortex in human volunteers, 80 ms after a small target moved 5 degrees horizontally from the centre of a VDU screen. Saccadic eye movements were recorded and experimental trials were compared with control, unstimulated trials. Magnetic stimulation was triggered in 70% of the trials selected randomly. The main effects of stimulation were: increased divergence of the eyes before each saccade, greater latency of saccade onset, and a tendency to undershoot the target. These results support the hypothesis that the posterior parietal cortex is involved in the programming of accurate saccades to visual targets.


Hypertropia following insertion of inferiorly sited double-plate Molteno tubes. The use of double-plate Molteno tubes is well established in the management of intractable glaucoma, particularly in eyes that may have had previous surgical procedures, often involving the superior conjunctiva with subsequent subconjunctival scarring. As it is frequently the case that the area of least scarring is inferiorly, we have sited the tubes there but have found that, although this usually provides good control of intraocular pressure, successful drainage of aqueous has been associated with the development of hypertropia due to upward displacement of the globe by fluid encapsulated around the plates. We report a retrospective study of 16 eyes undergoing inferiorly sited double-plate Molteno tubes, in which 9 eyes developed significant hypertropia at a mean time after insertion of 3.5 months. Mean degree of hypertropia was 9.8 prism dioptres. The management of this complication is discussed.


A comparison of the OKP visual field screening test with the Humphrey field analyser. In order to determine the loss of retinal sensitivity detectable by oculokinetic perimetry (OKP), we tested 27 eyes of 27 persons with glaucoma and 32 eyes of 32 patients with ocular hypertension with the OKP screener and with a central threshold test on the Humphrey analyser. The threshold at eight locations on the OKP chart between 12.5 degrees and 15 degrees from fixation was compared with the corresponding Humphrey perimetric thresholds. Seventeen eyes from 17 patients with glaucoma failed the OKP screening test by not seeing the target at one or more locations. The mean light sensitivity threshold at points seen on OKP screening was -5.8 dB (SD 4.6 dB) from aged-matched normals (AMN), whereas points missed had a mean threshold -16.1 dB (SD 9.3 dB) from AMN. The sensitivity and specificity with which OKP testing differentiated normal points from abnormal ones in Humphrey testing was 82.5% and 80% respectively, with a threshold criterion of -12 dB from AMN. Ten eyes from 10 patients with glaucomatous defects and all of the ocular hypertensive eyes gave normal responses on the OKP screening test. The glaucomatous eyes that passed were characterized by less severe defects on the Humphrey than those who failed in terms of mean deviation (0.02 < p < 0.05) or corrected pattern standard deviation (0.01 < p < 0.02). Four of the glaucomatous eyes to pass had a nasal step as the primary field defect. None of those to fail failed only on points within 10 degrees of fixation. We suggest further modification of the OKP screening chart to improve its efficiency.


Sezolamide: additivity to timolol twice daily. Sezolamide, a potent topical carbonic anhydrase inhibitor previously known as MK-417, was studied to determine its ocular hypotensive activity in patients with elevated intraocular pressure while on continuing therapy with topical timolol. This was a three-centre, double-masked, randomised, placebo-controlled, parallel study in 36 patients with bilateral primary open angle glaucoma or ocular hypertension on therapy receiving 0.5% timolol twice daily, with a morning intraocular pressure greater than or equal to 22 mmHg in both eyes 2-4 hours following an 8 a.m. dose of timolol. Sezolamide 1.8% or placebo twice daily was added to treatment with timolol on the evening of day 1 and continued for 2 weeks. Twelve-hour diurnal curves were performed before the study on day 1 (timolol alone) and on day 15. Intraocular pressure measurements were also taken on days 2 and 8 at 8 a.m. and 9 a.m. Patients who received timolol and sezolamide showed additional intraocular pressure reductions from day 1 (timolol alone) of 8.0 to 15.5%, which were significant at all times. At hours 1, 2, 4 and 8 the reductions in intraocular pressure observed in the group receiving sezolamide and timolol were significantly greater than those in the group receiving timolol and placebo.


Deterioration in the accuracy of the pulsair non-contact tonometer with use: need for regular calibration. The Pulsair non-contact tonometer (Keeler Pulsair: Keeler UK) has been shown to be a versatile instrument particularly suitable for screening for raised intraocular pressure. Although demonstrated to be accurate initially when compared to the Goldmann applanation tonometer no study has examined its long-term accuracy. Comparisons of three Pulsair tonometers with different degrees of usage with the Goldmann tonometer are described. Measurements were obtained from 64, 116, and 223 eyes in three separate comparative studies. Correlation coefficients of between 0.78 and 0.90 were obtained, the least used instrument being significantly more accurate than the two more extensively used instruments. Taking the Goldmann tonometer as the standard tonometer, and the aim to detect intraocular pressures of greater than 21 mmHg, sensitivities of 40%, 48%, and 85% for the three Pulsair tonometers respectively were shown. The Pulsair tonometer appears liable to a long-term drift in accuracy with use, and we suggest that provision is made for the regular re-calibration of the instrument.


Neurotoxic effect of vincristine on ultrastructure of hypothalamus in rabbits. The neurotoxic side-effects are a striking accompaniment of the therapy with vincristine (VCR). Data concerning the influence of VCR on the central nervous system are controversial. In the present study a schedule of VCR treatment was employed to develop the neurotoxic side effects in New Zealand rabbits. In all treated animals neurons were primarily and most severely affected cells. Most of them underwent degeneration. The consecutive stages of the increasing condensation of chromatin and cytoplasm of these cells were observed. Degeneration of the synaptic connections and secondary changes in myelin sheaths of degenerating axons were found. Debris of some death cells were localized intracellularly or as typical apoptotic bodies ingested by some glial cells. Astroglia underwent swelling and their processes separated dying neurons from the surroundings. All the results lead to conclusion that the VCR treatment concomitant with neurotoxic symptoms may affects regions of CNS lacking of blood-brain barrier. Neurons are most vulnerable cells of the brain parenchyma. The changes induced in neurons by the drug are characteristic in many aspects to those described in many cells undergoing apoptosis.


Effect of constant magnetic field on the liver of guinea pig. Electron microscopic studies. Twenty guinea pigs were exposed 1 hour daily, for 3 to 7 weeks to constant magnetic field (CMF), the induction of which was 0.005 T, and 0.3 T. Hepatocytes were examined in semithin sections with light microscope and TEM. The negative photographic plates of the TEM were analyzed and measured with densitometer. It was shown that CMF of the induction 0.005 T and 0.3 T exhibited structural changes in hepatocytes, primarily in mitochondria.


The monoclonal antibody (MAB) CD 68 allows the immunocytochemical identification of macrophages in primary and metastatic brain tumors in paraffin embedded tissues. While using the MAB CD 68 applied to paraffin embedded tissue specimen, macrophages could be marked in 31 primary brain tumors of glial origin and in 17 metastatic tumors from which 7 were carcinomas of the lung, 5 carcinomas of the breast, 2 from the digestive tract and 3 were clear cell carcinomas of the kidney. Despite quantitative differences between the primary and metastatic tumors in all specimen examined macrophages were found. The macrophages were localized within the tumor tissue, at the periphery of the tumor and its surroundings. In some cases cells with an intensive staining reactions were visible in blood vessel walls. No correlation between the type of tumor and intensity of macrophages infiltration has been found, however there were quantitative differences between primary and metastatic tumors.


Genetic changes in Morris hepatoma 7777 cells. Analysis of genetic changes in Morris hepatoma 7777 is described. Three different approaches were applied: DNA and chromosome transfection, karyotype analysis, and Southern hybridization modified by use of PFGE. Changes in the genome of tested cells were identified by PFGE and chromosome transfection. This supports the statement that PFGE is a useful method in transformed cell genome analysis.


A model of Ph' positive chronic myeloid leukemia-blast crisis cell line growth in immunodeficient SCID mice. A human Philadelphia-chromosome positive chronic myeloid leukemia-blast crisis (CML-BC) cell line BV173 proliferated in the hematopoietic tissues, infiltrated various organs and caused the death of immunodeficient SCID mice. Leukemia spreading was assessed with diminished number of bone marrow cells and caused splenomegaly. The leukemic colonies grew from single cell suspension of bone marrow, spleen and peripheral blood. Bcr-abl m-RNA was detectable in bone marrow, spleen, peripheral blood, liver, lungs and brain. Dying mice demonstrated severely hypoplastic bone marrow, splenomegaly and massive metastases in the liver and kidneys. The survival time of animals was dependent on the number of inoculated leukemia cells.


Anchorage-dependent growth factor(s) produced by rat sarcoma (XC) cells. The autocrine growth factor(s) was isolated from serumfree conditioned medium of rat sarcoma (XC) cells. Autocrine activity was enriched by ultrafiltration using Amicon YM 10 membrane, extraction with 1 M acetic acid and partially purified (650-fold) by chromatography on Bio-Gel P-100 and P-60. The final recovery of the autocrine factor(s) was 4 micrograms from 1800 ml of the conditioned medium (a yield of 6%). The factor(s) with molecular weight 6-10 kDa was heat and acid stable but inactivated by trypsin and dithiothreitol. It stimulated anchorage-dependent (but not anchorage-independent) growth of XC cells as well as untransformed, established lines of rat (NRK) and mouse (3T3) cells. The results obtained may suggest that autocrine factor(s) produced by XC cells can be one of EGF-like or/and insulin-like growth factors.


Selection of permethrin resistance in the malaria vector Anopheles stephensi. The laboratory strain of Anopheles stephensi, a well-known urban malaria vector, was selected with permethrin, a synthetic pyrethroid at LD90 level up to five generations. The selection resulted in the development of resistance in F5 generation to the tune of 13-fold to permethrin and cross-resistance to the tune of 7-fold to cypermethrin, 9-fold to alphamethrin, and 10-fold to deltamethrin. The development of cross-resistance to 4% DDT was also noticed. The susceptibility status against 5% malathion was maintained throughout the five generations. The synergistic effect of piperonyl butoxide with permethrin did not overcome the development of resistance. The development of resistance showed a significant relationship between hatchability and different generations.


Hepatic changes in P. falciparum malaria. Liver function tests were performed in 165 hospitalized patients suffering from P. falciparum malaria with complications. Serum bilirubin was found increased in 33 patients, and 22 of them had unconjugated hyperbilirubinaemia. Serum alanine aminotransferase was increased in 5 patients, but only to mild to moderate levels. Serum alkaline phosphatase was increased in 11 patients, gamma-glutamyl transpeptidase in 3 patients. Serum total protein and albumin were significantly decreased but these were considered more as indicator of acute phase response. Liver cell necrosis was observed in one patient, and oedema and mononuclear cell infiltration in two patients. Though hepatomegaly and mild elevation of enzymes can be observed in a significant proportion of patients, involvement of liver leading to acute hepatitis or liver cell necrosis is a relatively uncommon complication in P. falciparum malaria.


A longitudinal study to monitor chloroquine-resistant P. falciparum malaria in Bokajan and Manja PHC areas of Karbi Anglong District, Assam. In-vivo as well as in-vitro studies were carried out at regular intervals from 1979 to 1988 to monitor chloroquine resistance in P. falciparum in Bokajan and Manja PHC of Karbi Anglong district, Assam. The results showed no significant change in resistance status in the area. Intensified antivector measures, withdrawal of chloroquine pressure and prompt detection and treatment of P. falciparum cases with alternative drugs helped bring down the higher grades of resistant parasites by 1984, which however could not be maintained. The results of in-vitro tests correlated well with those of in-vivo tests. The Karbis seemed to have better immunity against the strain of P. falciparum than the non-Karbis residing in the area. However, there was no difference in the rate of sensitive and resistant cases detected amongst the two groups.


A comparative study of a new food supplement, ViviScal, with fish extract for the treatment of hereditary androgenic alopecia in young males. A controlled, randomized, double-blind, parallel-group study compared the effects of ViviScal (a new food supplement incorporating special marine extracts and a silica compound) with those of a fish extract in the treatment of young males with hereditary androgenic alopecia. The pretreatment histological diagnosis was alopecia with a mild to moderate perifollicular inflammation zone. The study consisted of 20 subjects who received two tablets of ViviScal once daily and 20 who received two tablets of fish extract once daily for 6 months. The mean patient age and mean duration and severity of baldness compared well between the two groups. Most patients had been treated with long-term topical 2% minoxidil for 1 year or more prior to the study. At baseline and after 6 months' treatment, a biopsy was taken for histological examination. A non-vellus hair count was performed at baseline and after 2, 4 and 6 months. In the fish extract treatment group three patients withdrew from the study before the fourth month due to lack of therapeutic effect. After 6 months' treatment, patients receiving ViviScal showed a mean increase in non-vellus hair of 38% compared with a 2% increase in the fish extract treatment group (P < 0.0001). In the ViviScal group, 19 (95%) subjects showed both clinical and histological cure, whereas none treated with fish extract showed any clinical or histological difference after 6 months' treatment (P < 0.0001). In both groups, a minimal decrease in the erythemal index was observed. In conclusion, ViviScal appears to be the first highly active treatment for androgenic alopecia in young males.


The effect of a single oral morning dose of nizatidine and ranitidine on intragastric pH under basal conditions and after pentagastrin stimulation. A comparison was made of the antisecretory activity of orally administered nizatidine and ranitidine by measuring intragastric pH under basal conditions and during and after pentagastrin stimulation. Intragastric pH values were measured with a bipolar glass electrode in 10 patients with healed duodenal ulcers treated with nizatidine or ranitidine according to a randomized single-blind design. The antisecretory activity of the two drugs was similar during the 4 h of monitoring following drug administration. Nizatidine, however, showed a more rapid inhibitory action than ranitidine, producing a significantly greater increase in pH with respect to basal values during pentagastrin infusion. In the period following infusion the pH values observed with ranitidine were higher than with nizatidine, but not significantly so. Under these experimental conditions, therefore, the antisecretory activity of nizatidine was shown to be more rapid than that of ranitidine and equally effective.


In vitro evaluation of plasticizer activity on the growth and metabolism of chick embryo fibroblasts and on the development of chick embryo lungs. Administration of di(2-ethylhexyl) phthalate (DEHP) to primary cultures of chick embryo fibroblasts brought about a decrease in cell proliferation rate after 48 h and an inhibition of both DNA and protein synthesis measured by [3H]thymidine and [3H]leucine, respectively, after 48 h. The growth of chick embryo lung rudiments in vitro was also depressed by DEHP treatment. Lung rudiment were smaller in DEHP-treated embryos after 6 days' treatment. These results indicate that DEHP has a cytostatic effect on embryonic cells and tissues.


Effects of nicergoline on calcium and magnesium deposition in the central nervous system tissues of rats maintained on low-calcium diets. Reduction of calcium intake leads to the mobilization of calcium and magnesium from the bone pool and to calcium deposition in the soft tissues, especially in the central nervous system (CNS). The effects of 10 alpha-methoxy-1,6-dimethylergoline-8 beta-methanol 5-bromonicotinate (nicergoline), an ameliorator of cerebral circulation and metabolism, on the deposition of calcium and magnesium in the CNS, heart, liver, kidney, muscle, abdominal aorta and bones were studied in rats maintained on standard and low-calcium diets. Rats were fed the following diets for 90 days: standard calcium (12.5 g/kg); standard calcium with 60 mg/kg nicergoline; low-calcium (30 mg/kg); and low-calcium with 60 mg/kg nicergoline. The presence of nicergoline did not affect blood chemistry but magnesium concentrations in the liver were significantly (P < 0.05) higher in rats fed standard diet with nicergoline. Magnesium concentrations in the occipital cortex, pons, cerebellum, liver, kidney, muscle and femur of nicergoline-treated rats fed low-calcium diet were significantly (P < 0.01-0.05) higher compared with those in the corresponding controls, whereas the calcium concentrations in the femur of nicergoline-treated rats fed both standard and low-calcium diets were significantly (P < 0.05) higher than those in the corresponding controls. In general, nicergoline tended to preserve the calcium content in the bone of rats fed a standard diet. Nicergoline may be implicated in calcium metabolism in rats fed low-calcium diets and may activate cerebral metabolism through the maintenance of magnesium concentrations in the CNS and soft tissues.


Agranulocytosis during treatment with fluconazole. Fluconazole, a new effective triazole antifungal agent, has been reported to cause fewer adverse reactions than amphotericin B. A patient who was diagnosed as having agranulocytosis and thrombocytopenia after treatment with fluconazole was investigated and recovered after withdrawal of the antifungal therapy. This case suggests the need for careful haematological observation during the treatment with fluconazole.


Development of a recombinant vaccinia-rabies vaccine for oral vaccination of foxes against rabies. To improve both safety and stability of the vaccines used in the field to vaccinate foxes against rabies by the oral route, a recombinant vaccinia virus, expressing the immunizing G protein of rabies virus (VVTGg RAB-26D3 187 X P strain) has been developed. The c-DNA corresponding to the glycoprotein of the ERA strain of rabies virus has been inserted into the thymidine kinase (TK) gene of the vaccinia virus (Copenhagen strain). The efficacy of this recombinant strain was tested by the oral route, primarily in foxes. The duration of immunity conferred by the VVTGg RAB, a minimum of 12 months in cubs and 18 months in adult animals, corresponds to the length of protection required for fox vaccination in the field. VVTGg RAB innocuity was tested in foxes and in domestic animals as well as in numerous wild animal species that could compete with the red fox in consuming vaccine baits in Europe. During a minimum of 28 days post vaccination, neither clinical signs nor lesions were observed in any of the vaccinated animals. Moreover no transmission of immunizing amounts of the recombinant occurred in the red fox or the other species tested. To study the stability of the vaccine strain, baits containing the vaccine were placed in the field. Despite considerable variations of environmental temperatures, the VVTGg RAB titre remained stable after one month in the field. Since all the baits are taken within one month, it can be assumed that most of the animals taking the baits are effectively vaccinated.(ABSTRACT TRUNCATED AT 250 WORDS)


Rabies vaccines for veterinary use: difficulties in establishing potency acceptability thresholds. The variability of a control test is sometimes difficult to assess. In fact this variability should be considered as a common basis of any biological test. The example of the NIH or PER test is given for veterinary rabies vaccines. The same acceptability threshold is accepted by everybody, both producers and control laboratory. But biological variability should induce the producers to determine a "working acceptability threshold" higher than the conventional one. This safety margin should be determined by the producer to counterbalance the variability of the test both in the producer and control laboratories so that most batches submitted to the official control are accepted. On the other hand, control laboratories should fulfil two "fair" requirements: reveal their flow charts and their margin of error to allow the producer to make the needed adjustments; reduce as far as possible the uncertainty of control tests by using the most accurate statistical interpretation to allow the producers to establish a "working acceptability threshold" not too much higher than the official one.


Control methods and thresholds of acceptability for antibrucella vaccines. Protection against brucellosis involves both cellular and humoral effectors not yet fully appreciated. Living or killed vaccines can protect against the infection itself or only against abortion. For official controls, vaccines (or new procedures of vaccination) must first be characterized pharmacologically and tested for innocuity. Protection must be tested on natural hosts with a reference vaccine (S19 or Rev. 1) by the agreed method which reproduces the natural infection and measures immunity in toto. Control and vaccinated females are challenged by the conjunctival route at mid-pregnancy under standard conditions (strain, dose) to measure the resulting infection by bacteriological analysis of excretion at parturition and of infection in target organs at slaughter. Results are principally expressed by the infection rate which should be +/- 95% in the control group. In the new vaccine group the rate should be equivalent to, or lower than, the reference vaccine group. To be statistically valid, at least 30 animals per group are required. For routine controls, laboratory models using guinea pigs, not well standardized, inaccurate and expensive, have long been proposed. The mouse model, extensively studied and standardized, should now be preferred to the guinea pig model. In the mouse model, residual virulence of a living vaccine is estimated by the time required by 50% of the mice to eradicate the strain from their spleen (Recovery Time 50%). Immunogenicity is measured by the ability of mice to restrict their splenic infection after a virulent i.p. challenge at a dose (B. abortus 544; 2 x 10(5) cfu) chosen in order that all mice were still infected 15 days post challenge.(ABSTRACT TRUNCATED AT 250 WORDS)


Establishing an acceptability threshold for equine influenza vaccines. Shortcomings in the original methods (based on haemagglutination of erythrocytes) used to measure potency of equine influenza vaccines and antibody responses stimulated by vaccines, coupled with the lack of a reliable challenge system in the target species, has hindered progress in identifying the antigenic content required to provide protection. Reliable methods are now available for measuring the haemagglutinin (HA) content of vaccines and the antibody responses they elicit. The development of challenge systems in the target species has allowed antibody levels consistent with protection to be identified under experimental conditions. Comparison of vaccine performance under experimental conditions and in the field suggests that exposure to a nebulised virus aerosol provides a realistic challenge for the assessment of equine influenza vaccines.


Control of manufacture--principles and purposes. The control of manufacture is centred on the assessment of the operation based on information supplied by the manufacturer and on detailed physical inspection. The purpose of controlling manufacture is to ensure, as far as practicable, that products are manufactured within premises, and by processes and personnel, of such standard that the quality of the final product is assured. The required standards are written in the "Guide to Good Manufacturing Practice for Medicinal Products", and its several supplementary guidelines, so in practical terms the purpose of inspection is to assess compliance with these guidelines. It is usual, however, for manufacturers' premises and practices not to comply with the guidelines in all respects. There is therefore a need to make judgements on the significance of, and risks associated with, these discrepancies. It is necessary to consider potential problems in the context of the overall manufacturing process, bearing in mind the nature of the products involved. A decision as to what remedial actions are required, and over what time-scale, is then made.


Inspection of veterinary biologics in the United States. Inspection of veterinary biologics in the United States (U.S.) has changed significantly over the years since the Virus-Serum-Toxin Act (V-S-T) was passed by the U.S. Congress in 1913. At first, government inspectors were appointed to every production location, observing the preparation, testing and initial shipment of veterinary biologics. In the 1950s, a new plan was developed to provide a more scientific basis of inspection. Over a 10-year period, a programme to test finished products at the National Veterinary Services Laboratories and a new procedure for inspecting facilities and products was implemented. Our current programme retains our extensive pre-licensing review of products, labeling, facilities and personnel and adds the new inspection system of random checking of finished batches, unannounced in-depth inspections, control of product batches and post-licensing monitoring. This combination of activities ensures that pure, safe, effective and economical veterinary biologics are available for use in the U.S.


Licensing veterinary biologics in the United States. The authority for the regulation of veterinary biologics in the United States is provided in the Virus-Serum-Toxin Act, enacted in 1913 and amended in 1985. The Act authorizes the Secretary of Agriculture to prescribe regulations governing the preparation and marketing of veterinary biologics shipped into, within, or from the United States. The veterinary biologics program carries out the mandate to ensure that all veterinary biological products under the U.S. jurisdiction are pure, safe, potent, and efficacious. The program is based on licensing, inspection, and testing. Establishment licensing defines the production facilities, manufacturing practices, and the responsible person. Product licensing defines the product, the method of production, and testing requirements; it also assures product purity, safety, potency, and efficacy. Biologics production is based on the Master Seed concept, in which a master parental stock is the source of all seed materials for production. The final product may not be more than five serial passages removed from the Master Seed. Host animal immunogenicity is demonstrated by statistically valid host animal vaccination and challenge studies using the minimum level of antigen at the highest level of passage. Potency tests are correlated to host animal immunogenicity and are conducted on each serial of product prior to release for marketing to assure efficacy. Conditional licensing procedures that assure purity, safety and a reasonable expectation of efficacy are used to provide products for emergency conditions, limited markets, local situations, or other special circumstances. Licensing for further manufacturing permits two or more licensees to work together in the production of a product.(ABSTRACT TRUNCATED AT 250 WORDS)


Control of product batches (before and after registration). The German approach. At present, batch control is performed after a product is licensed. The range of batch control tests and the detailed description of these tests are fixed for every product during the respective authorization procedure. In an increasing number of cases it also seems necessary to test the prelicensing batches, which are used in the clinical trials for efficacy and safety. The batch control performed in the Paul-Ehrlich-Institute consists of the control of the batch protocols and the practical control of product samples. The practical testing includes purity, efficacy and safety of the batch. Considerable quality differences occur from one batch to another, even when the batch-to-batch-consistency is documented during the authorization procedure. Moreover, without previous indication to the state authority, the authorized control tests are frequently modified by the producer or are not performed for each batch. Some examples of these occurrences are presented. In addition it is necessary to bear in mind that the production of vaccines and sera needs biological seed materials, the quality of which can never be so uniform in the way chemical substances normally are. Since most of the incidents which occur when using vaccines and sera are batch-related and not product-related, the continuous batch control by state authorities is considered to be necessary.


Control of product batches (before and after registration). The British approach. In the United Kingdom, some pre-registration testing of immunological products is carried out. However, this is normally not extensive. Usually the tests are laboratory tests undertaken to verify results obtained by the manufacturer or quality controller. For many years, routine post-registration testing of batches of immunological products has been carried out. However, this has ceased and had been replaced by a new system known as the Quality Assurance/Quality Control (QA/QC) scheme. The new scheme is designed to provide the Veterinary Medicines Directorate (VMD) with assurance of the continuing quality of batches of immunological products. The basis of the scheme is a system of inspection of companies' quality control facilities and procedures, every eight months, by staff with appropriate microbiological qualifications and experience. Before an inspection is carried out, the company must provide detailed documentation on the facilities and the quality control procedures used for each product. This provides the inspector with the background information for the inspection and discussions with the company.


Control of product batches (before and after registration). The French approach. The issue of investigation into vaccine files and control in general has always been a delicate one. There are considerable economic and medical interests. The supervisor should not then thwart a step towards progress and should not appear as an economic brake. Yet he should guarantee to the users a certain quality in the marketed biological products. According to these principles, recommendations have been formulated to indicate tests that should be realized by manufacturers and related in files of request for Marketing Authorization. Consequently the level of requirements may seem quite high in France. But partners involved in the investigation of files and in vaccine controls as well as those who are concerned with the marketing of new biological products should guarantee their quality. In addition to investigating the file, controls are undertaken in France by State laboratories which specialize in a particular species. They are also applied research laboratories. These controls aim at checking the manufacturers' results. In France no systematic control is undertaken on successive vaccine batches before marketing. Controls are undertaken at random if information coming from users gives rise to suspicion about a vaccine or batch of vaccines which are causing problems. However systematic controls should be undertaken by a private or State laboratory for imported batches. In future, only control results will probably be required. However batch-to-batch controls are undertaken by a state laboratory for rabies vaccines and tuberculin. In conclusion, in France we favour controls before Marketing Authorization together with high specifications for manufacturers. State batch-to-batch control is not undertaken since present means do not encourage such a procedure, the interest of which are not obvious; furthermore, this procedure remains very restricting for manufacturers.


Control of product batches (before and after registration). The Belgian approach. A review is given on the registration procedure and controls of veterinary vaccines in Belgium. An obligatory registration procedure was installed in 1969, and has since been modified several times. The Belgian approach for control of veterinary vaccines before and after registration is explained. Controls before registration are often carried out exclusively by the manufacturer and are described in the registration file. Controls may, however, also be carried out by an independent laboratory if the manufacturer so desires. In general, the procedures described in a national or European Pharmacopoeia must be applied if a monograph is available. Batch control after registration is carried out by the manufacturer. When manufactured in Belgium, controls on the finished product must also be carried out by the Institute of Hygiene for viral vaccines and some selected bacterial vaccines (e.g. anthrax). In addition, all batches of imported vaccines are subject to controls both by an officially accepted laboratory and by the Institute of Hygiene, the first to be paid by the importer, the second free of charge. Batch controls include homogeneity of the batch, labelling, storage conditions and also identity of active component(s), safety, sterility, absence of extraneous agents and potency. The potency test described in the original registration file must be used for batch control.


Testing of veterinary biologics in the United States. The National Veterinary Services Laboratories (NVSL) in Ames, Iowa, are responsible for the confirmatory testing of veterinary biologics produced in the United States and regulated by the United States Department of Agriculture. There were 22.101 serials of biological products produced and submitted to the NVSL during fiscal year (FY) 1991 (October 1, 1990, to September 30, 1991). All serials were tested by the manufacturer; and 2.086 (9.44 percent) were also tested by the NVSL for purity, safety, or potency of at least one component. Of the serials tested, 97.84 percent were found to be satisfactory. The total number of tests conducted during FY 1991 was 4.283. The criteria for establishing test rates and test methods are discussed. In addition to the testing of finished biological products, the NVSL tested 129 bacterial or viral master seeds and 31 master cell stocks during FY 1991 for identity and freedom from extraneous bacterial and viral agents. These master seeds and master cell stocks will be used to make future biological products.


The cost of quality assurance/quality control. Good Manufacturing Practice Guidelines have an inherent philosophy to maintain high standards and to promote uniformity within trading areas. Reaching these standards in some countries and maintaining them in others is costly. The Cost of Quality is regarded as a significant indicator of business performance. Quality costs are increasing as demands for improved accuracy and precision are made on the industry. The paper also details actual costs for validating a modern biological production facility and establishing biological quality control facilities. Bench mark quality control testing costs for a typical large and small animal vaccine are given.


The future European system for the free circulation of veterinary vaccines in Europe. Although Community legislation has only recently been extended to cover veterinary biologicals, these products are fully covered by the recent Commission proposals for the free movement of medicinal products after 1992. The proposals are based on more than 25 years of harmonization of national regulations and more than 10 years' experience of coordinating national decisions on individual medicinal products. The proposals envisage the establishment of two new Community authorization procedures: a centralized procedure based on a single scientific evaluation of the highest possible quality to be undertaken within a new European Medicines Evaluation Agency, leading to a binding EEC-wide decision, with the possibility of limited exceptions to take account of differences in disease status in the Member States; a decentralized procedure based on the concept of mutual recognition of national decisions, with binding Community arbitration in the event of a dispute. To provide the Community institutions with the scientific and technical resources necessary to operate these new procedures, the proposals also provide for the establishment of a European Medicines Evaluation Agency.


Presentation of the EEC directive (81/852)--quality. The EC directive 81/852 has been revised to include a number of parts setting out the information to be included in applications for marketing authorizations for veterinary immunological products. Part 6 contains the details of the quality aspects which must be addressed by the applicant. It is divided into sections and provides the applicant with the structure and layout to be used for the presentation of the information in the dossier. The applicant must provide sufficient detail to allow the competent authority to assess the quality of the product, including aspects such as batch-to-batch consistency and the risk of contamination of batches with extraneous agents. Thus the dossier must contain details of the starting materials, the method of manufacture, and the controls and tests on the starting materials and intermediate and finished products. The details of the blending and composition must be included. Limits of acceptance should be provided. Procedures used such as inactivation and purification steps and quality control tests should have been shown to be appropriate and the results of the validation studies must be presented. The information supplied must be accompanied by results obtained from batches manufactured by the method described. A justification of the proposed shelf-life of the product must be made with supporting evidence from real-time stability studies.


Presentation of the directive 81/852/EEC (92/18) safety testing. The aim of the directive is to define tests to be done by a manufacturer which are capable of providing as precise information as possible about the properties of the biological products concerning safety. These tests have to be done in the target species and the dose used should be that quantity of the product recommended for use and containing the maximum titre of potency. The samples for safety testing should be taken from a batch produced according to the manufacturing process. The vaccine is tested in normal conditions at the recommended dose and by each recommended route of administration to animals of each species and category it is intended to vaccinate. Objective criteria have to be recorded to measure possible adverse general and local effects. But tests in abnormal conditions of use may be necessary, such as administration of an overdose or repeated administration of one dose. Effects on reproductive function and progeny have to be studied as well as investigations which may be required in some cases to evaluate a possible adverse effect on the immunological function of the vaccinated animal. Special requirements seem to be appropriate for live vaccines: to evaluate the spread of the vaccinal strain from vaccinated to unvaccinated animals including, if necessary, wild species; to determine whether the vaccinal strain can disseminate in the vaccinated organism; to check for the absence of reversion to virulence of live stains especially for conventional vaccines. Field studies can therefore provide complementary information about safety.


Current USDA procedures for licensing biotechnology-derived veterinary biologicals. The establishment of appropriate procedures for regulating the commercialization of biotechnology products is an international issue which has been debated extensively in many countries. This paper will discuss the current procedures that have been established for the regulation of biotechnology-derived veterinary biologicals within the United States Department of Agriculture (USDA) and more specifically within Biotechnology, Biologics, and Environmental Protection (BBEP) of the Animal and Plant Health Inspection Service (APHIS). These procedures implement recent BBEP regulatory policy that has been developed to ensure risk-based regulation and avoid excessive restrictions that might curtail the development of biotechnology products.


The American manufacturer's view on the EEC texts and harmonization. In a world that has become increasingly smaller with the aid of modern air travel, computers and facsimile machines, the European Community's efforts toward harmonization are applauded by the Animal Health Institute, representing the major U.S. manufacturers of veterinary biological products. The new and proposed directives and guidelines are based on the latest technology and help ensure that only high-quality safe and effective veterinary biologicals are registered in the European Community. These new legislations are, however, significantly different from the current United States (USDA) licensing requirements. The USDA requirements are designed to ensure the safety and efficacy of veterinary biologicals licensed in the United States and have been effective in doing so. Among major differences between EEC and USDA requirements are the EEC requirements for GLP safety tests, GMP for manufacturing, with increased measures for the prevention of cross-contamination, duration of immunity data, and raw material specifications in excess of those required in the United States. Very tight deadlines for implementing these requirements have been established. In the spirit of international harmonization and with the goal of reducing or preventing potential trade barriers, the AHI would like to propose the formation of an international working group consisting of FEDESA, AHI, CVMP, USDA and other international industry and government officials. It is suggested that annual meetings be held in conjunction with either FEDESA or AHI meetings. Issues to be dealt with include standardization of test requirements, time lines for implementation and other issues that could reduce or prevent trade barriers without adversely affecting product quality.(ABSTRACT TRUNCATED AT 250 WORDS)


The role of the Office International des Epizooties in the standardisation of biologicals. The Office International des Epizooties (OIE) is the World Animal Health Organisation created in 1924 in Paris. The following activities have been started in order to promote standardisation of biologicals: a) preparation of the OIE Manual of Standards for Diagnostic Tests and Vaccines; b) organisation of Reference Laboratories for diseases of Lists A and B; c) establishment, in cooperation with WHO, FAO and IAEA, of international standards for diagnostic tests with particular reference to the ELISA. The Manual will be distributed worldwide in 1992 and 1993 and this will contribute to an internationally unified approach to diagnosis and immunoprophylaxis, in other words to international harmonisation of veterinary biologicals. It is planned that future OIE Reference Laboratories, now being organised, will supply the necessary standards.


Regulation of Drosophila neural development by a putative secreted protein. The Drosophila strawberry (sty) locus was isolated by P-element insertion mutagenesis in a screen for mutations affecting eye development. Analysis of the mutant phenotype and the putative expression pattern of the sty gene suggested that it has multiple functions. Mutations in the sty gene lead to irregular spacing of ommatidia, an increase in the number of photoreceptor cells, as well as abnormal axonal projections to the lamina and disrupted structure of the optic lobes in the adult fly. The sty mutation also causes abnormal head involution, a change in a number of sensilla in the antennomaxillary complex in the embryonic stage and abnormal morphogenesis of the maxillary palp and wings in later stages. We examined the presumptive expression of the sty gene during development by histochemical staining for lacZ expression from enhancer trap elements inserted within the sty gene. During embryogenesis, expression of lacZ showed a segmental pattern in the ectoderm and in the nervous system. In the eye imaginal discs, lacZ was expressed in photoreceptor cells beginning a few rows posterior to the morphogenetic furrow. The lacZ was also expressed in the wing disc. In the adult, lacZ was expressed in the retina and lamina. We cloned the sty gene by P-element tagging and found that it encodes a putative secreted protein containing a cysteine-rich region similar to the epidermal growth factor (EGF) repeat. On the basis of the loss of functional phenotype, the expression pattern and the predicted structure of its product, we propose that sty encodes a diffusible protein acting as a signal involved in lateral inhibition within the developing nervous system and also as a factor involved either directly or indirectly in axonal guidance and optic lobe development.


Modulation of the invasive phenotype of human colon carcinoma cells by organ specific fibroblasts of nude mice. We examined whether fibroblasts from subcutaneous, colon or lung tissues of nude mice influence the invasive potential of highly metastatic human colon carcinoma KM12SM cells. Primary cultures of nude mouse fibroblasts from skin, lung and colon were established. Invasive and metastatic KM12SM cells were cultured alone or with fibroblasts. Growth and invasive properties of the KM12SM cells were evaluated as well as their production of gelatinase activity. KM12SM cells were able to grow on monolayers of all three fibroblast cultures but did not invade through skin fibroblasts. The conditioned media of KM12SM cells cocultured with skin, colon or lung fibroblasts were examined for the presence of type IV collagenase (gelatinase). KM12SM growing on plastic and on colon or lung fibroblasts produced significant levels of latent and active forms of 64 kDa type IV collagenase, whereas KM12SM cells cocultivated with nude mouse skin fibroblasts did not. In contrast, human squamous cell carcinoma A431 cells produced significant levels of collagenase type IV when cocultured with nude mouse skin fibroblasts, a tissue they invaded and completely penetrated. Incubation of KM12SM cells in serum-free medium containing recombinant human interferon-beta (fibroblast interferon) was associated with significant reduction in gelatinase activity. Since the production of type IV collagenase by human colon cancer cells is specifically inhibited by mouse skin fibroblasts but not by colon or lung fibroblasts the data suggest that organ-specific fibroblasts can influence the invasive and metastatic properties of KM12SM cells.


Differential and stage dependent effects of retinoic acid on chondrogenesis and synthesis of extracellular matrix macromolecules in chick craniofacial mesenchyme in vitro. Retinoic acid (RA) is well known to be a potent teratogen and induces a variety of facial defects in vivo, but at concentration levels lower than those that cause facial defects, RA seems to play an important role in normal facial development. In a previous study, we demonstrated the ability of RA to stimulate chondrogenesis in vitro in HH stage 23/24 chick mandibular (MND) but not frontonasal (FNP) mesenchyme cultured in a serum-free medium. The present study furthers these results by examining the effects of RA on chondrogenesis of chick facial mesenchyme at earlier embryonic stages and the effects on cell proliferation and synthesis of specific extracellular matrix macromolecules at stage 23/24. MND and FNP cells were cultured as micromasses for 4 days in defined media. As described previously, chondrogenesis in stage 23/24 MND cells was significantly enhanced by concentrations of RA of 0.1-1 ng/ml; however, at all earlier stages examined (18 to 22) RA at these concentrations had no significant effect. Higher concentrations of the retinoid inhibited chondrogenesis in MND cultures from all stages tested. Cells of the FNP from all stages displayed no significant change in chondrogenesis below 1 ng/ml RA and a dose dependent inhibition at higher concentrations. Thus RA's promotional effects in the face are not only tissue specific (MND), but also stage-dependent (HH 23/24). The specific effects of RA on matrix production and cell proliferation of stage 23/24 MND and FNP cells was examined by analysis of 35S sulfate, 3H thymidine and 3H proline incorporation. Analysis of 35S sulfate incorporation into sulfated proteoglycans confirmed that concentrations of RA of 0.1-1 ng/ml stimulated cartilage matrix production in MND but not FNP cultures. Above this level of RA, 35S sulfate incorporation was reduced in both. Likewise, 3H proline incorporation into collagenous protein, and to a lesser extent non-collagenous proteins, was stimulated by low levels of RA in MND, but not FNP cultures. Higher concentrations of the retinoid in either MND or FNP cultures did not lower collagen production, undoubtedly due to stimulation of non-chondrogenic cells within the population. This indicates that levels of RA as high as 100 ng/ml cause phenotypic change rather than cell death. This last point is corroborated by the analysis of 3H thymidine uptake in the cultures which was only transiently modified in most. The data indicate that cell proliferation occurred even in the presence of high RA levels.


Identification of a short nuclear lamin protein selectively expressed during meiotic stages of rat spermatogenesis. The nuclear lamina is a karyoskeletal structure located at the nuclear periphery and intimately associated with the inner nuclear membrane. It is composed of a multigene family of proteins, the lamins, which show a conspicuous cell type-specific expression pattern. The functional role of lamins has not been definitively established but available information indicates that they are involved in the organization of nuclear envelope and interphase chromatin. Spermatogenesis is characterized, among other features, by stage-specific changes in chromatin organization and function. These changes are accompanied by modifications in the organization and composition of the nuclear lamina. In previous experiments we have determined that rat spermatogenic cells express a lamin closely related, if not identical, to lamin B1 of somatic cells; whereas rat somatic lamins A, C, D and E were not detected. Considering that chromatin reorganizations during spermatogenesis may be directly or indirectly related to changes of the nuclear lamina we have decided to further investigate lamin expression during this process. Here we report on the identification of a 52 kDa protein of the rat which, according to immunocytochemical and biochemical data, appears to be a novel nuclear lamin. Using meiotic stage-specific markers, we have also demonstrated that this short lamin is selectively expressed during meiotic stages of spermatogenesis.


Distribution of hyaluronan in the mouse endometrium during the periimplantation period of pregnancy. The tissue distribution of stromal hyaluronan (HA) in the periimplantation mouse uterus was studied histochemically using a biotin-labelled HA-binding complex from cartilage proteoglycan. HA is present around proliferating stromal cells in both the pregnant and pseudopregnant mouse uterus prior to their differentiation into the decidualized phenotype. Decidualization is accompanied by clearance of HA from the extracellular matrix (ECM). This clearing is part of an intrinsic developmental program of the differentiating deciduum. A specific embryonic signal from the implanting conceptus is not required for this phenomenon to occur, since a similar response could be induced in deciduoma produced by artificial stimulation of a receptive uterus. Clearing of HA from the antimesometrial stroma is consistent with the hypothesis that the HA-negative decidual cell may be involved in restricting the invasion of trophoblast cells during embryo implantation. Retention of HA within angiogenic regions of the decidua basalis implies a functional role for this molecule in placental vascularization.


Developmental regulation of class I major histocompatibility complex antigen expression by equine trophoblastic cells. Between days 36-38 of pregnancy equine trophoblastic cells of the chorionic girdle migrate and form endometrial cups. Just prior to invasion, the chorionic girdle cells express high levels of polymorphic, paternally inherited, major histocompatibility complex (MHC) class I antigens. Their descendents, the mature, invasive trophoblast cells of the endometrial cups, however, express low or undetectable levels of MHC class I antigens by day 44 of pregnancy. Experiments with MHC compatible pregnancies, the study of residual chorionic girdle cells that had failed to invade the endometrium and remained on the surface of a conceptus, and the study of chorionic girdle cells recovered on days 34-36 of pregnancy and then maintained in vitro for up to 24 days strongly suggest that the reduction of MHC class I antigen expression by mature invasive trophoblast cells of the endometrial cups is developmentally regulated. This phenomenon does not appear to be induced by a maternal antibody response or by other uterine factors acting after the chorionic girdle trophoblast cells invade the endometrium.


Ascorbate independent differentiation of human chondrocytes in vitro: simultaneous expression of types I and X collagen and matrix mineralization. In this study we describe the collagen pattern synthesized by differentiating fetal human chondrocytes in vitro and correlate type X collagen synthesis with an intracellular increase of calcium and with matrix calcification. We show that type II collagen producing fetal human epiphyseal chondrocytes differentiate in suspension culture over agarose into hypertrophic cells in the absence of ascorbate, in contrast to chicken chondrocytes which have been shown to require ascorbate for hypertrophic differentiation. Analysis of the collagen synthesis by metabolic labeling and immunoprecipitation as well as by immunofluorescence double staining with anti type I, II or X collagen antibodies revealed that type X collagen synthesis was initiated during the third week. After 4 weeks culture over agarose we identified cells staining for both type I and X collagen, indicating further differentiation of chondrocytes to a new type of 'post-hypertrophic' cell. This cell type, descending from a type X collagen producing chondrocyte, is different from the previously described 'dedifferentiated' or 'modulated' types I and III collagen producing cell derived from a type II collagen producing chondrocyte. The appearance of type I collagen synthesis in agarose cultures was confirmed by metabolic labeling and immunoprecipitation and challenges the current view that the chondrocyte phenotype is stable in suspension cultures. An increase in the intracellular calcium concentration from 100 to 250 nM was measured about one week after onset of type X collagen synthesis. First calcium deposits were detected by alizarine red S staining in type X collagen positive cell nodules after 4 weeks, again in the absence of ascorbate. From these observations we conclude a sequence of events ultimately leading to matrix calcification in chondrocyte nodules in vitro that begins with chondrocyte hypertrophy and the initiation of type X collagen synthesis, followed by the increase of intracellular calcium, the deposition of calcium mineral, and finally by the onset of type I collagen synthesis.


[Genetic relationships between European and Transcaucasian mice of the genus Apodemus Kaup]. Genetic relations between four European mice species of the genus Apodemus (Apodemus sylvaticus, A. flavicollis, A. microps and A. falzfeini) and five Transcaucasian ones (A. mystacinus, A. microps, and the forms No 2, 3p, 3f) were studied for 37 biochemical loci. Close genetic relations were demonstrated between the mice of subgenus Sylvaemus and A. mystacinus. A. microps and A. falzfeini from the Caucasus were shown conspecific to these species from the Ukraine and the presence of the two new different species--the form No 2 and 3p in the Caucasus was established.


[Molecular-genetic analysis of tandem repeats in intron 6B of the CFTR gene in various populations and in cystic fibrosis patients]. Frequencies of hexa- and heptatandem tetrameric repeats in Russian slavonians from the North-Western part of the country were found to be 0.19 and 0.81, respectively, with the frequency of heterozygotes estimated as 0.31. Similar values of allelic frequencies were found for uzbek and azerbaijan populations (0.24 and 0.76; 0.22 and 0.78, respectively). These data assess the existence of genetic equilibrium of both alleles in the populations studied. Hexa TTR is present twice more often in the CF chromosomes than hepta TTR common for normal chromosomes. Absolute genetic disequilibrium of both TTR alleles was observed for CF chromosomes bearing the F508 mutation. The latter was exclusively detected in a conjunction with hexa TTR allele. Combined application of the F508 test and TTR alleles assay significantly increased the number of totally informative CF-high risk families, and thus might be highly beneficial for a subsequent prenatal diagnosis of CF.


[Structure of phenotypic variability in readiness potential in 6-7 year-old children]. This report is a part of a larger project, the major purpose of which is a longitudinal study of developmental changes in genetic control of variation in Bereitshaft potentials (BP). 15 pairs of MZ and 15 pairs of DZ twins aged 6-7 years participated in the BP study. 2 second epochs of EEC time-locked to the movement were digitized off-line at 500 Hz (DAS-16G "Metrabyte"), for 1500 msec, prior to and 5000 msec after movement. A complex BP waveform with the positive components was discovered in young children. We fitted four basic models to the twin data for each of the BP in F3, F4, C3, C4. Since the models (VA, EW) in F3, F4, C3 and (EB, EW) in C4 fit very well and cannot be improved markedly by adding the most important alternative parameters, we conclude that variation of BP of F3, F4, C3 is best explained by a very simple genetic model, and variation of BP of C4 is best explained by a simple environmental model.


[Molecular nature of beta-thalassemia in Tajikistan: a four base pair deletion in codons 41-42 of the beta-globin gene]. Thirty tajiks, whose relatives had beta-thalassemia traits (revealed in previous investigations by determination of the HbA-2 and HbF levels) were selected to screen beta-thalassemia mutations. DNA samples from each individual were subjected to the PCR (polymerase chain reaction) to amplify the 635 bp beta-globin gene fragment. One additional band was detected in three samples after the amplified fragment underwent electrophoresis in 2% agarose gel and the EtBr was stained, and two additional ones were revealed by 6% PAAGE and staining of the EtBr. All additional bands migrated more slowly than appropriate 635 bp fragment. It is supposed that additional bands are heteroduplexes formed from the wild type chains and mutated chains carrying a deletion or insertion. The 4 bp deletion of the 41-42 (-tctt) was detected after the direct sequencing of the amplified fragments. This mutation is common among Chinese but it was not revealed in the Middle Asia populations. The mutation can be easily screened using the PCR and electrophoresis in 2% agarose gel or PAAG of the amplified beta-globin gene fragments.


[Mapping of the focus of action of the lethal allele of the ecslt76 gene using genetic mosaics]. Using gynandromorph the fate map of Drosophila melanogaster blastoderm was constructed. The focus of the lethal allele lt76 of the ecs gene was localised in two map sites. The site localized in the anterior part gives rise to the anterior region of the nervous system, and head. The latter covers the blastoderm anlages which are involved in the development of abdomen, and the genital imaginal disc. Interaction between two systems controlling morphogenesis is under debate.


[Effect of mei mutations on the processes occurring in the double super-unstable system at the yellow and scute loci in Drosophila melanogaster]. The influence of meiotic mutations on the mutation changes in the double super-unstable system in the yellow and scute loci of Drosophila melanogaster was studied. The mei-41D5 and mei-218 mutations changed the spectrum and frequency of mutagenesis in males of the y2nsscme strain, in contrast to the postulate that meiotic mutations do not interfere with male recombination in D. melanogaster. These mutations also changed the frequency and spectrum of mutagenesis in females. In particular, they inhibited mutagenesis at early stages of ovogenesis. Meiotic conversion did not change specifically by mei mutations. At the same time, the mei-41D5 mutation increased all recombination processes in meiosis. The results obtained indicated the involvement of genetic recombination in mutation changes occurring in the double super-unstable system. Therefore, the latter may be successfully used in studies of the role of different genes and their products in recombination.


[Genetic control of meiotic recombination in yeast]. A review of research on genetic control of meiotic recombination is presented. The genes controlling different stages of meiotic recombination were revealed. Possible relationship of the gene products with the process of genetic recombination is under discussion.


Recombination between the X and Y chromosomes and the Sxr region of the mouse. The Sxr (sex-reversed) region that carries a copy of the mouse Y chromosomal testis-determining gene can be attached to the distal end of either the Y or the X chromosome. During male meiosis, Sxr recombined freely between the X and Y chromosomes, with an estimated recombination frequency not significantly different from 50% in either direction. During female meiosis, Sxr recombined freely between the X chromosome to which it was attached and an X-autosome translocation. A male mouse carrying the original Sxra region on its Y chromosome, and the shorter Sxrb variant on the X, also showed 50% recombination between the sex chromosomes. Evidence of unequal crossing-over between the two Sxr regions was obtained: using five markers deleted from Sxrb, 3 variant Sxr regions were detected in 159 progeny (1.9%). Four other variants (one from the original cross and three from later generations) were presumed to have been derived from illegitimate pairing and crossing-over between Sxrb and the homologous region on the short arm of the Y chromosome. The generation of new variants throws light on the arrangement of gene loci and other markers within the short arm of the mouse Y chromosome.


Electrofusion of mouse embryos results in uniform tetraploidy and not tetraploid/diploid mosaicism. Some previous attempts to produce tetraploids experimentally have resulted in a proportion of treated embryos becoming 2n/4n mosaics at a frequency which may be as high as 20%, when using cytochalasin B as a fusigenic stimulus and cytogenetic techniques to identify putative tetraploid embryos. To investigate the possible occurrence of 4n/2n mosaicism, tetraploid embryos were produced by electrofusion, a process which allows adjacent blastomeres at the 2-cell stage to fuse following exposure to electric field pulses. Embryos used for electrofusion were hemizygous for a transgene consisting of approximately 1000 copies of the mouse beta-globin gene. After in situ hybridization, one hybridization signal is expected per diploid genome. Tetraploid cells in 7.5-, 8.5-, 9.5- and 10.5-day-old conceptuses were distinguished from diploid cells by performing in situ hybridization on histological sections. The frequency of nuclei with two hybridization signals in the 'hemizygous' tetraploid embryos was compared to diploid embryos which were either hemizygous or homozygous for the beta-globin transgene. Comparison of the frequency of nuclei with two hybridization signals between tissues of 'hemizygous' tetraploid conceptuses and homozygous diploid conceptuses showed no significant difference, which implies that the tissues in the tetraploid conceptuses were uniformly tetraploid. No evidence was found to suggest that electrofusion results in 2n/4n mosaicism.


The time of detection of recessive visible genes with non-random mating. Expressions for the probability and average time of detection of a recessive visible gene in populations where there is partial selfing or partial full-sib mating are presented. A small increase in the proportion of inbred matings greatly reduces the average time until detection and increases the proportion detected. Unless the proportion of inbred matings or the population size is very small, the time and proportion detected are approximately independent of the population size.


Estimating effective population size from samples of sequences: a bootstrap Monte Carlo integration method. We would like to use maximum likelihood to estimate parameters such as the effective population size N(e) or, if we do not know mutation rates, the product 4N(e) mu of mutation rate per site and effective population size. To compute the likelihood for a sample of unrecombined nucleotide sequences taken from a random-mating population it is necessary to sum over all genealogies that could have led to the sequences, computing for each one the probability that it would have yielded the sequences, and weighting each one by its prior probability. The genealogies vary in tree topology and in branch lengths. Although the likelihood and the prior are straightforward to compute, the summation over all genealogies seems at first sight hopelessly difficult. This paper reports that it is possible to carry out a Monte Carlo integration to evaluate the likelihoods approximately. The method uses bootstrap sampling of sites to create data sets for each of which a maximum likelihood tree is estimated. The resulting trees are assumed to be sampled from a distribution whose height is proportional to the likelihood surface for the full data. That it will be so is dependent on a theorem which is not proven, but seems likely to be true if the sequences are not short. One can use the resulting estimated likelihood curve to make a maximum likelihood estimate of the parameter of interest, N(e) or of 4N(e) mu. The method requires at least 100 times the computational effort required for estimation of a phylogeny by maximum likelihood, but is practical on today's work stations. The method does not at present have any way of dealing with recombination.


Do Eve's alleles live on?

Do Eve's alleles live on? Consider a random sample of genes at a locus, drawn from a population evolving according to the infinitely many, neutral, alleles model. The sample will have a most recent common ancestor gene, which we shall call 'Eve'. The probability distribution, for the number of genes of oldest allelic type in a sample, is known and has a neat form. Rather less is known about the distribution for the number of genes in the sample which are of the same allelic type as Eve possessed. If the latter number is positive, then these genes are automatically of the oldest type in the sample. But Eve may have no non-mutant descendants in the sample; then, the oldest allele will be a mutant arising in a line of descent after Eve. The paper studies the number of non-mutant descendants from Eve, its distribution and moments. It seems that there may be few neat results. In large samples, the proportion of genes of Eve's type has an approximate beta-like density, together with a discrete probability atom at zero, if the mutation rate parameter is low. Extinction of the allele of even the population's common ancestor is possible, but not certain, and bounds are obtained for its probability. Some comments are made about the applications and implications of the results for human mitochondrial DNA.


Changes in superoxide dismutase and catalase in aging heat-shocked Drosophila. We have examined the age-dependent expression of CuZn superoxide dismutase (SOD) and catalase (CAT) in Drosophila melanogaster following heat shock. Quantitative northern blot analysis was performed after heat shock on 2-, 23- and 49-day-old flies, using a 0.48 kb Sal1 EcoR1 fragment of the Drosophila SOD cDNA and a 1.4 kb fragment of the human catalase cDNA. Heat shock induction was monitored with a 5.4 kb DNA fragment of the Drosophila hsp70 gene. After exposure to 37 degrees C for 30 min and 60 min, the level of SOD RNA in young flies was elevated above that of nonstressed conditions. Changes in the transcription of SOD gene with age paralleled the expression of hsp70 RNA. The SOD RNA was elevated in heat-shocked middle aged (23-25 days old) flies compared to young Drosophila (2 days old), then it decreased in 49-50-day-old flies. The relative expression of CAT RNA did not change with age or after heat shock. Enzymatic activities of these two antioxidant enzymes were evaluated in nondenaturing polyacrylamide electrophoresis gels. SOD migrates on this gel as three different electromorphs. These were designated as fast, intermediate and slow migrating bands. The most intense activity was associated with the fast band in these flies. In the absence of heat shock, there was an age-dependent decrease in the intermediate, but not in the slow or fast bands. Heat shock does not affect the intensity of the fast band in young or old flies, however; in middle aged flies, there is a shift in this band toward the slow position. No change was detected in the activity of catalase with age or heat shock, although flies of all ages exhibited a shift toward a faster-migrating electromorph with increasing time of heat shock. This effect was also observed in flies fed H2O2 and is more pronounced in insects fed higher concentrations. These results are discussed in relation to the role of these antioxidant enzymes in protecting against age-induced oxidative stresses.


[Evaluation of central speech disorders using objective criteria: a psychophysiologic contribution to the discussion of the neural plasticity hypothesis]. Examinations of aphasic patients by using cognitive tasks were based on the hypothesis that semantically evoked potentials correlate to the processing of information in the different speech processing of information in the different speech processing areas. It was found that patients with Broca aphasia generated synchronization potentials in the Wernicke area in contrast to Wernicke patients. We suppose a correlation between the timing in sensorial speech processing areas and the generation of synchronization potentials.


[Dysphonia following strumectomy with normal respiratory movement of the vocal cords]. Voice function was phoniatrically investigated pre- and postoperatively in 66 patients undergoing thyroidectomy. Postoperatively we found a disturbance of the voice in 25 patients (43%), though there was a normal function of the recurrent laryngeal nerve. Typical symptoms were a reduced average pitch of the speaking voice, a lowering of the upper and the lower limit of the range of pitch, a lower volume and a fast fatigue of the voice. Eight times the voice remained reduced, even 6 months postoperatively. The cause is not seen in a neural lesion but in a disturbance of the prelaryngeal musculature. The frequency of the voice changes emphasizes the importance of the phoniatric examination, especially in patients who need their voice professionally.


Characterization of the electroglottographic waveform: a primary study to investigate vocal fold functioning. To objectify the vocal fold functioning we opted for a method of processing the electroglottographic (EGG) signal. This study with 7 normal and 12 pathological voices presents the results of measurements over the chest register as a function of voice intensity and frequency. Reproduction measurements show that at least 5 measurements per intensity/frequency pair are needed to get a reasonable estimation of the EGG parameters. The averaged results over about 20 measurements in the chest register showed differences for some parameters for subjects in the most different categories. To get more consistent results we recommend to improve the measurement protocol and adjust the parameters.


Transitional determinacies.

Transitional determinacies. In classic generative grammar a distinction is drawn between linguistic 'competence' and linguistic 'performance', the former referring to linguistic knowledge, the latter to how linguistic knowledge is used. However, this controversial differentiation obscures the additional dichotomy between linguistic knowledge for production and linguistic knowledge for recognition. In this article it is shown that production and recognition differ, that recognition is not simply the inverse of production, and that the derivation of production from recognition and recognition from production require a small set of generalizable 'transitional determinacies'. Secondly, it is shown that transitional determinacies explain the difference between 'overt' and 'covert' recognition recently observed in prosopagnosics, patients unable to recognize familiar faces. Prosopagnosics and normals are found to differ in their transitional determinacies, such that prosopagnosics require more binders (precisors) for covert recognition than normals. In general, it is concluded that transitional determinacies are as necessary to the theory of grammar as determinacies themselves.


Sound spectra in cleft palate patients with a Sanvenero-Rosselli and modified Honig secondary velopharyngeal flap. The averaged sound spectra produced by cleft patients with a Sanvenero-Rosselli (SR, n = 4) and modified Honig (MH, n = 4) velopharyngeal flap were compared with each other and with the spectra produced by 4 healthy controls. Each subject produced 16 sound spectra; the total number of spectra was 432. The results indicated that the averaged sound spectra produced by MH patients resembled more closely the spectra produced by healthy controls, while the spectra by SR patients differed from those of MH patients and controls. It was concluded that the differences in the averaged spectra of SR and MH patients reflected differences in the region of the oral cavity, particularly the oropharynx rather than the region of the nasopharynx. The results of this preliminary study indicate that this topic is worth further studies.


Perceptual comparison of neoglottal, oesophageal and normal speech. The sex, normality, intelligibility, rate, rhythm and intonation of 44 neoglottal, oesophageal and normal speakers have been judged by a panel of 10 trained listeners. It was found that the sex of the alaryngeal speakers was perceived correctly much less reliably than that of the normal speakers. Neoglottal speakers were rated more normal and intelligible than oesophageal speakers. The speaking rate of neoglottal speakers was judged not to be significantly different from that of normal speakers. Neoglottal speakers were considered more fluent and to have better intonation than the other alaryngeal speakers. Thus neoglottal speakers were found, on average, to be as good as or better than good oesophageal speakers in each of the respects of which judgements were made.


[Importance of the EEG in early and differential diagnosis of dementia of the Alzheimer type]. The development of new parameters in the quantitative evaluation of EEG provides new diagnostic opportunities for the clinician. Reviewing all published papers on EEG and dementia over the last 10 years revealed 30 out of 428 studies discussing EEG alterations in relation to the severity of the disease. This approach provides useful information for early and differential diagnosis of dementia. Besides a generalised slowing-down represented by an increase of delta and theta and a decrease of alpha and beta in the frequency spectrum (also characterised by a reduction of the peak frequency), the topography of the alterations changes depending on the severity of the disease. For instance, the alpha activity loses its normally occipital orientation. In early stages of the disease, it is measured parietally ("anteriorized"). In advanced stages, the activity is equally distributed over the scalp. Some studies found significant alterations even in early stages of the disease, most pronounced in theta and beta activity, making the EEG a useful tool for the early diagnosis of dementia. But studies on the differential diagnostic value are lacking. The value of EEG could be improved after parameters like the EEG segmentation or the traditional parameters combined with cognitive stimulation have been sufficiently evaluated.


[Electroencephalographic findings in dementia diseases]. Even if generally the EEG cannot yield specific pointers that are typical for a special pattern of dementia, it is nevertheless meaningful and helpful to perform routine EEG in demented patients. EEG is the functional additional examination that does not place any stress on the patient and is easy to perform at a low cost (1). As a functional diagram the EEG reflects the electrical processes taking place in the cerebral cortex, yielding an excellent image of the cerebral functional state. Assessment of the EEG is done in accordance with internationally standardised criteria. Particular attention is directed at a slowing-down of the background activity, the occurrence and extent of scattered theta and delta waves, their topographical distribution and the development of pathological EEG characteristics during recording (44). Update spectral analysis EEG procedures, i.g. the possibility to calculate power spectra for the individual EEG frequency bands and their topographic distribution, it may be possible in the near future to further improve differentiation between normal and pathological EEG findings. The functional dynamic aspect of the EEG is its particularly strong point in the differential diagnostic clarification of dementia patterns. By repeated EEG recordings, i.e. by close follow-up, and by relating the electrophysiological findings to the clinical pattern, reliable pointers are obtained for the extent of the activity of the process underlying the dementia pattern. EEG is predestined like no other method to enable early detection of rapid changes in cerebral function thanks to its easy operation and unlimited repeatability. The occurrence of parenrhythmic theta and delta waves in the EEG reflects in a special manner the acuity of an organic basic process. In this way EEG enables differentiation between acute and chronic processes of cerebral damage. Over and above this, correlation with the relevant clinical findings makes it possible to differentiate between reversible states of dementia and irreversible defect syndromes and hence also to point to the long-term outcome. Finally, the occurrence of certain patterns of findings or the identification of characteristic wave forms allows essential differential diagnostic pointers and definitions.


[Neurologic concomitant diseases in Crohn disease]. Disturbances of the central and peripheral nervous system in Crohn's disease can be directly or indirectly caused by the disease itself or by the treatment. The first mentioned disturbances are very seldom. Cerebral arterial and venous thromboses can be attributed to hypercoagulation. Malabsorption of vitamin B 12 or folic acid can lead to a subacute combined degeneration of the spinal cord, sometimes in combination with a polyneuropathy and an encephalopathy. Spinal abscesses very seldom occur. An opticusneuropathy can be caused either by vasculitis or by a lack of vitamin A and/or vitamin B. Polyneuropathies in Crohn's disease which are not induced by drugs are manifested as mononeuritis multiplex or as symmetrical sensory neuropathy. An autoimmune process is being discussed as the probable cause of there diseases. In some cases large doses and prolonged administration of metronidazole can lead to cerebral dysfunctions with state of confusion, alterations of consciousness, cerebral convulsions and cerebellar syndrome. Most of these symptoms disappear rapidly after this drug is not longer administered. In long-term administration of metronidazole 10-50% of the patients develop a sensory polyneuropathy with a total dosis of at least 22.5 g, but mostly above 60 g. After the use of metronidazole is stopped, it takes a substantial period of time until there is a full recovery from the symptoms.


Noninvasive approaches to pain control in terminal illness: the contribution of psychological variables. Pain is a common problem for patients with terminal illnesses. The major efforts to control pain for these patients have focused on the appropriate uses of analgesic medication to reduce the sensory aspect of the pain experienced. There are a number of studies in the chronic pain literature, however, that have implicated the important role of a number of psychological factors in the maintenance and exacerbation of pain. Among the most important variables identified are expectancy, anxiety, perceived controllability, self-efficacy, symptom preoccupation, operant conditioning, classical conditioning, and observational learning. In this paper, these psychological variables are examined and their applicability to patients who have pain associated with terminal illnesses is discussed as well as the implications of these variables for complementary treatment with analgesic medications are described.


Pain relief using cutaneous modalities, positioning, and movement. Positioning, movement, and certain cutaneous modalities may be easily used by all caregivers, including the family, to bring comfort and pain relief to terminally ill patients with pain. For such patients, these techniques are most appropriately used in addition to pharmacologic control of pain. Patients themselves may use some of the cutaneous modalities with minimal assistance from others, thereby promoting a sense of independence. Other techniques may be performed by family and friends, providing them with the assurance that they are assisting a loved one. The techniques presented here can be readily used in the home or hospital setting and are relatively low risk, simple, and inexpensive. This paper presents specific guidelines for patients and caregivers in relation to the use of superficial massage, superficial heat and cold, menthol application to skin, transcutaneous electrical nerve stimulation (TENS), positioning, and movement. Because of their simplicity and ease of use, these techniques tend to be overlooked. However, taking the time to introduce these techniques to patients and families often results in a significant contribution to the comfort of the dying patient.


Involvement of families in pain control of terminally ill patients. Pain associated with terminal illness is an extremely stressful problem for the patient as well as family members. Helping family members cope increases their effectiveness as caregivers and improves their own quality of life. Conversely, improving the patient's ability to cope decreases stress on other family members. Optimal treatment of pain in the terminally ill may be conceptualized and administered from a family perspective. Methods for helping families of terminal pain patients cope are organized around a theme of enhancing feelings of control and self-efficacy. Control over pain and related problems may be maximized by providing education, improving decision making and assertiveness skills, and by teaching specific techniques for pain and stress management, including proper analgesic use, progressive relaxation, imagery, distraction techniques, and time management. These techniques are described from a family perspective in this paper.


The role of patient education in cancer pain control. Patient education should be a central component of pain control regimens for cancer patients. Few systematically developed and carefully evaluated pain control patient education programs have been reported. Patient education for cancer pain control should include five phases: assessment, goal setting, selection of educational strategies, implementation and reassessment. Each of these phases should be included to maximize the goals of pain prevention and pain relief.


Facilitating the use of noninvasive pain management strategies with the terminally ill. In this paper a number of issues that appear to transcend the range of noninvasive interventions described in this volume are examined. Specifically, we raise the important issue of barriers to treatment. Among those discussed are patients', their significant others', as well as health care providers' conceptualizations of and biases regarding pain and pain control. We emphasize that it is important not only to focus on the details of the specific techniques described but also on how most effectively to present information. We note that it is important to customize any noninvasive intervention to the specific needs of the patient and their significant others. We provide some suggestions as to how this might be accomplished. Finally, we discuss some attitudes and beliefs held by health care providers that may inhibit their use of the full range of noninvasive techniques that are available for managing pain.


Cancer pain: a multidimensional perspective. Cancer pain has traditionally been studied from a unidimensional/medical model. However, limitations of the medical model have led researchers to the development of multidimensional models of cancer pain. The current manuscript describes a multidimensional model of cancer pain with six components: physiological, sensory, affective, cognitive, behavioral and sociocultural. Research relevant to each component of the model is reviewed with an emphasis on future research directions.


Assessment of the terminally ill patient with pain: the example of cancer. Efforts to understand pain associated with terminal illness have been guided traditionally by the biomedical model in which psychological and environmental factors are considered incidental and not causally significant influences of pain. More recent conceptualizations of pain, however, recognize that pain can be affected by a variety of factors including mood, beliefs about pain, past learning, as well as physical perturbations. This development has led to assessment strategies that are more comprehensive, multidimensional, and less singularly aligned with a biomedical model. The greatest amount of attention to pain among the terminally ill has focused on cancer patients. Thus, in this paper we will describe a comprehensive, multi-dimensional assessment of cancer pain. Information regarding cancer and cancer pain is first presented and then a strategy for comprehensively assessing cancer pain is outlined. Recent developments in the assessment of cancer pain are briefly reviewed.


The cognitive behavioral perspective on pain management in terminal illness. Cognitive Behavioral Therapy (CBT) can be an effective non-medical treatment for the alleviation of pain-related suffering in patients with advanced disease. CBT offers a common-sense theoretical model and a variety of rational and pragmatic techniques that are easy for both patients and health professionals to understand and practice. The perspective emphasizes two ideas: that pain and suffering are not the same, and that patients can exercise substantial personal control over their thoughts, feelings, and behavior. Suffering is a feeling of pervasive personal destruction, whereas pain is a sensory stimulus that does not necessarily imply general personal disintegration. CBT teaches patients to practice relaxation and active coping skills to increase pain tolerances, and to modify thoughts and attitudes that increase the perception of personal disintegration. Even in the most advanced stages of a terminal illness patients can practice these skills and experience themselves as active agents that can reduce their suffering and enhance their sense of well-being.


Hypnosis and related techniques in pain management. Hypnosis has been used successfully in treating cancer patients at all stages of disease and for degrees of pain. The experience of pain is influenced not only by physiological factors stemming from disease progression and oncological treatment, but also from psychosocial factors including social support and mood. Each of these influences must be considered in the successful treatment of pain. The successful use of hypnosis also depends upon the hypnotizability of patients, their particular cognitive style, their specific motivation, and level of cognitive functioning. While most patients can benefit from the use of hypnosis, less hypnotizable patients or patients with low cognitive functioning need to receive special consideration. The exercises described in this chapter can be successfully used in groups, individual sessions, and for hospice patients confined to bed. Both self-hypnosis and therapist guided hypnosis exercises are offered.


Engineered antibodies as pharmacological tools. The ability to engineer the antibody molecule has now progressed to the stage where one can realistically contemplate creating a pharmacologically useful targeting molecule comprising a single-chain, minimal antibody combining site together with a domain that imparts a second functionality. It will certainly be possible in the near future to adjust the fine specificity and affinity of the antibody domain, either by in vitro selection methods or by site-directed mutagenesis based on structural criteria. The functional domain could be an enzyme, a toxin, or any other protein that suits a pharmacological purpose. In this review I have illustrated these directions primarily by summarizing work from my laboratory and those of my collaborators. The review covers single point mutations in the heavy chains of digoxin-specific antibodies that either strikingly diminish or strikingly enhance recognition of a small feature of the antigen, the position 12 OH moiety in the steroid nucleus. The principles for constructing a minimal, single-chain antigen-binding domain based on one of the digoxin-specific antibodies are also outlined, as are the principles for incorporating such domains into fusion proteins. Finally, as a practical application of antibody-targeted enzymes, the construction and evaluation of an anti-fibrin antibody-single-chain urokinase fusion protein is examined. This protein has enhanced potency and specificity both in vivo and in vitro, and it offers the promise of increased therapeutic efficacy as well as diminished toxicity.


Engineering antibodies for therapy.

Engineering antibodies for therapy. Success in the generation of an antibody-based therapeutic requires careful consideration of the binding site, to achieve specificity and high affinity; of the effector, to produce the desired therapeutic effect; of the means of attachment of the effector to the binding site; production of the end product; and the response made by the patient to the administered compound. Each of these areas is receiving attention by antibody-engineering techniques. The number of potentially useful monoclonal antibodies developed over the last 10 years, and currently in clinical trials or preregistration, is now being increased by these engineered newcomers. It will be interesting to see over the next few years how many of these antibodies, and of which kind, emerge as products.


Therapeutic human antibodies derived from PCR amplification of B-cell variable regions. Despite advances in the in vitro immunization of human B cells (Borrebaeck et al. 1988) and the development of immunodeficient mice (McCune et al. 1988) for the reconstitution of the human immune system ex vivo, immortalization of antigen-specific human B cells remains the limiting step in the generation of human monoclonal antibodies. Typically this is performed with the aid of Epstein-Barr virus transformation followed by subcloning, confirmation of antigen binding and hybridization of the B lymphoblasts to a suitable fusion partner such as GLI-H7. This general approach is effective and widely used; however, it is time-consuming with erratic results. These were the immediate reasons we and others devised methods to directly obtain the variable regions from small numbers of human B cells (Larrick et al. 1987). The success of the PCR-based approach is illustrated above. In the present studies we successfully captured and stably produced antibodies from the V regions of two potent human anti-tetanus antibodies secreted by heteromyelomas that were too unstable for scale-up production. Although further preclinical evaluation of these antibodies is in progress, results to date indicate that the recombinant antibodies produced in myeloma-based cell lines or CHO cells are equivalent in binding specificity and activity to the native heteromyeloma-derived antibodies. Recent studies from this laboratory indicate that effective anti-tetanus protection will require a cocktail of anti-tetanus antibodies. Details of this work will be the subject of a future communication (Lang et al., in preparation).