Effects of ricinoleic and oleic acids on the digestive contractile activity of the canine small and large bowel. Oleic and ricinoleic acids or their trans isomers, elaidic and ricinelaidic acids, were administered intraduodenally and evaluated for their effects on the digestive motor activity of the canine small and large bowel. Four dogs of mixed breed were implanted with duodenal cannulas and extraluminal strain gage transducers along the circular axis of the proximal and mid-jejunum, terminal ileum, and proximal colon. After feeding 200 g of canned dog food to induce continuous contractile activil of isotonic saline was given into the duodenum as a single bolus infusion and recordings were made for a 1-hr period. A 30-ml volume of saline served as control. Administration of each cis fatty acid produced an initial stimulation in jejunal areas of about  a 2-min duration followed by a post-stimulatory inhibition. Both the initial stimulation and post-stimulatory inhibition were greater for ricinoleic acid than for oleic acid. Minimal or no effects were produced in ileal or colonic areas. In contrast, the trans isomers produced little or no effect on either the small or large bowel. Alterations in the digestive contractile patterns produced by oral administration of 10 ml of oleic, ricinoleic acid or their respective triglycerides were also tested. Triolein had no effects. Ricinoleic acid and castor oil poduced a brief initial stimulation followed by polonged inhibition of small bowel motor activity. Both cathartics had a mild laxative effect. Digestive motor patterns returned to control approximately 45 min after oleic acid. There was no indication at any time of an initiation of continuous contractile activity after ricinoleic acid or castor oil which could justify the use of the terms irritant of stimulant to describe their actions.


Billiary lecithin secretion. II. Effects of dietary choline and biliary lecithin synthesis. Choline which is absorbed from the intestine isne produced no change in bile salt pool size or secretion rate. However, with each increment in dietary choline, biliary lecithin as well as cholesterol secretion rates were significantly increased. The results demonstrate that although bile salt is required for biliary lecithin secretion, the magnitude of secretion is principally dependent upon the extent of biliary lecithin synthesis, which in turn may be regulated by the amount of dietary choline. Results further indicate that biliary cholesterol secretion is more closely linked to lecithin than to bile salt secretion. These findings would therefore suggest that lecithin secretion might be independently manipulated when bile salt is deficient to achieve enhanced biliary cholesterol transport.


Development of a Crohn's disease activity index. National Cooperative Crohn's Disease Study. Needing a single index of degree of illness in Crohn's disease, the National Cooperative Crohn's Disease Study group collected data prospectively from 187 visits of 112 patients with Crohn's disease of the small bowel, colon, or both. Information on 18 predictor variables was gathered at each visit. In addition, the attending physician rated his over-all evaluation of how well the patient was doing and compared the patient's status with that at the previous visit. A multiple regression computer program was utilized to derive an equation for prediction of the physician's over-all ratings from a subset of the predictor variables fulfilling a combination of constraints. This equation, numerically simplified and utilizing eight selected variables, is the Crohn's Disease Activity Index. Index values of 150 and below are associated with quiescent disease; values above that indicate active disease, and values above 450 are seen with extremely severe disease.


Serum lysozyme in inflammatory bowel disease. Serum lysozyme activity was measured in 18 patients with ulcerative colitis and 40 patients with Crohn's disease by both the turbidimetric and lysoplate method. Only one patient with ulcerative colitis and eight patients with Crohn's disease had increased serum lysozyme activity by either or both methods. Both methods appeared equally sensitive in detecting increased serum lysozyme activity. In Crohn's disease, the percent with elevated values increased with increase in disease activity.


Catecholamine-containing nerve fibres in the human abdominal vagus. The vagal nerve of man has been investigated for the presence of adrenergic nerve fibres using the histochemical fluorescence method of Hillarp and Falck. Following 30-60 min of nerve ligation during surgical operations, the right anterior main trunk (subdiafragmatic level) from one patient, and the anterior nerve of Latarget of 5 patients were found to contain unmyelinated nerve fibres with accumulations of green fluorescent material representing a catecholamine. The observations indicate the presence of adrenergic nerve fibres running caudally in the human vagal nerve, in accordance with similar findings in other mammals, e.g. cats and dogs.


[Is maternal mortality today still a problem? (Results of the statistics for the federal state of Hamburg from 1968 to 1972) (author's transl)]. This is the fourth review on the maternal mortality and obstetrics in the federal state of Hamburg comprising, this time, the years 1968 to 1972. 70 cases of maternal mortality are described classified, and discussed. All cases of maternal death with a temporal relationship to pregnancy, labour and delivery and puerperium were analyzed individually. There were 70 maternal deaths in 113, 564 deliveries. 10 or 8.8/100,000 cases were due to embolism. 1 or 0.9/100,000 cases were due to amniotic fluid embolism. 10 or 8.8/100,000 cases were due to infection. 9 or 7.9/100,000 cases were due to hemorrhage. 8 or 7.0/100,000 cases were due to toxemia of pregnancy. 5 or 4.4/100,000 cases were due to criminal abortions. 1 or 0.9/100,000 cases were due to chorio-carcinoma and 26 or 23/100,000 cases were due to non-obstetric causes. Our experience over many years shows that maternal mortality, stillbirth and death during the first year of life should be considered an epidemiologic entity.


[Bilateral radical mastectomy for carcinoma of the breast of the stages T0 and T1 (N0, M0) and mammoplasty of both breasts, areola and nipples in a single operation (author's transl)]. Bilateral radical mastectomy and concomitant mammoplasty is a new operative procedure for the treatment of carcinoma of the breast in the stages T0 and the small T1, N0, M0. The advantage of the procedure is the increased extent of the operation, the improved cosmetic result and the single stage of the procedure. Consequently the cure rate is improved and the psychic trauma to the patient is decreased. The operation is more radical since the so-called healthy breast is operated first, including axillary lymphadenectomy and subsequent mammoplasty followed by the same procedure on the so-called diseased breast. If the pectoral muscles are involved, a conventional radical mastectomy has to be done. The diagnosis is made by excisional biopsy and frozen section microscopy. The cosmetic result is improved because both breasts are subjected to the same operation, and lateral differences in shape, volume and consistency of the breasts are eliminated. Cylastic prostheses are inserted as new breasts usually sub-pectorally and rarely subcutaneously. The nipples are surgically reconstructed and pigmented by tattooing. The advantage of this single stage procedure is a single general anaesthesia and less psychic trauma since the patient goes to sleep with the knowledge of waking up with two breasts even though endoprothetic breasts. Post-operative radiotherapy can be applied with the prostheses in place. The principle of cure before cosmetics is preserved. Radical operation of the so-called healthy side is justified because of the above mentioned cosmetic advantages and the 25% incidence of primary or metastatic cancer of the so-called healthy side. The post-operative care of women with a bilateral operation is not a problem since no more breast tissue is left behind. Orthopedic complaints which can occur because of macromastia of the remaining breast are also eliminated.


[Life threatening infection with bacteroides fragilis in connection with Dalkon shield (author's transl)]. A case of severe peritonitis in connection with the application of a Dalkon Shield is reported. By culturing the peritoneal exsudate a monoculture of Bacteroides fragilis was found as causing agens. The general aspects of severe infection in connection with IUD, particularly the Dalkon Shield, are discussed. By analysing the reports in the literature it cannot be ruled out, that Bacteroides fragilis may to be a major factor in cases with lethal outcome.


[A comparison between so-called triple-diagnosis and surgical biopsy in diagnosis of the female breast (author's transl)]. A comparison of 265 cases of tripel-diagnosis and 112 surgical biopsies of the female breast is reported. In 89,83% of these 265 cases the correct diagnosis was found, in 9,43% the result was doubtful, in 0,75% completely wrong. In 3,77% of these doubtful results the repetition of the tripel-diagnosis gave the correct diagnosis, in 4,15% surgical biopsy we found 2,68% wrong diagnosis. This fact shows the certainty of tripel-diagnosis. In addition the highly significant lower rate of complications, the avoidance of the risk of narcosis and scars and the lower amount of costs show the high clinical value of the so-called tripel-diagnosis.


[Indications and results of antenatal karyotyping (author's transl)]. From 1972 to 1975, 64 genetic amniocenteses and 72 cell cultures from amniotic fluids were done for antenatal karyotyping. In 3 cases a therapeutic abortion for genetic indication was carried out. The indications for antenatal karyotyping, the technique and complications of genetic amniocentesis in early pregnancy and the methods of cell cultures of amniotic fluid are discussed. A compilation of the results of 1918 antenatal karyotypings reported in the literature shows the following probability for the presence of an unbalanced chromosomal anomaly in the fetus: in mothers over age 40 4.25%, in mothers age 35-39, 1.45%, in cases with trisomy-21 in a previous sibling 0.73%, in cases with balanced chromosomal anomalies in the parents, 15.2%. In the next few years an increase of antenatal karyotyping in the different risk groups can be expected. In the individual case the indication for genetic amniocentesis must be balanced against the risk of complications.


[Do cytological criteria exist to determine the correct technique of a cytosmear of the uterine cervix? (author's transl)]. The objective of this study was to determine whether the presence or absence of endocervical and/or metaplastic cells in a pap smear from the uterine cervix is indicative that the smear has been obtained from the correct location. Cytosmears from positive cytological findings were compared with cytosmears from correct negative and false negative findings for the presence of endocervical and/or metaplastic cell types. It was found that smears with positive findings showed no endocervical and or metaplastic cells in 25-30% of the cases of carcinoma in situ or dysplasia and in 75% of the cases of invasive carcinoma. False negative smears contained endocervical and/or metaplastic cells in 50% of the cases, although no atypical or dyskaryotic cells were detectable. The presence or absence of endocervical and/or metaplastic cell types in a cytosmear from the uterine cervix is therefore no indication that the smear has been taken from the correct location.


[Birth after a chorionepitheliom with pulmonary metastasis (author's transl)]. We report about a woman, 20 years old, who get a chorionepitheliom with pulmonary metastasis. We treated not by operation but by cytostatics Methotrexat and Actinomycin D over 40 days. The remission of the tumor was significant but accompanied by considerable toxic sideeffects.--18 months after this, we finished a new pregnancy by sectio caesarea without any complications for wife or child.


[Pregnancy and fetal outcome after cessation of oral contraceptives (author's transl)]. In a total of 4149 prenatal patients there were 849 patients who became pregnant following the cessation of the use of oral contraceptives. Results were compared with pregnancies of a similar series of 1000 patients who never had employed contraceptive steroids. Comparison shows a lower incidence of abortion of 5,6% in the oral contraceptive group, compared with 9,2% among the control group. Results show also a lower incidence of toxemia. The incidence of obstetrical surgery was a little higher than in the control group. The incidence of premature birth, perinatal mortality, congenital anomalies, the frequency of twins and of asphyxia neonatorum were similar in both groups. A special group of 107 pregnancies occurring immediatly after cessation of the pill shows no higher risks in pregnancy, during birth or with regard to fetal outcome. An exception is made by the incidence of premature birth that was in this special group 8,8% compared with 5,4% among the control group.


[Aspects to birth control (author's transl)]. From 1964, the year with the highest new born rate since 1945, until 1972, the number of births in the BRD was reduced for 34%. Simultaneously, the birth rate of women between 20 and 30 years was decreased for a third. Presently, the average age of half of the women who deliver their first or second child is over 30 years. While on the one hand there are investigations on the practical experiences which are hidden behind the obvious "one child effect", it would be, on the other hand, dangerous, to lay out a demographic "production policy" which shows no interest in economy. In birth-control, biotic-private individual foundations are variable elements; social values, however, are non individual objectives [corrected] with temporal relatively great constancy. The splitting of these elements is, in the 20th century, tracing back with violent actuality to the original big line of Malthus and Darwin, which is to maintain sufficient scope for nourishment and reproduction to the human world with its vegetable and animal life. This concerns industrial and development countries as well. The challenge is to line out acceptable ideas for the solution of the Circulus vitiosus, based on statistical data, on socio-philosophic understandings which have to be worked out yet and on common sense.


[The psychological results of tubal ligation as definitive method of contraception (author's transl)]. The cases of tubal ligation at the University Department of Tubingen and the University Department of Giessen during the last few years were reviewed. In most cases the psychological results of the procedure is positive and results in an improved partner relationship in most cases, provided that the procedure was the result of a mature decision by the couple. Tubal ligation in the puerperium is more favorable than in conjunction with the Caesarean Section. Interval sterilization shows the best results. A thorough discussion by the physician and if possible a psychological discussion should take place prior to the tubal ligation since both are important aids in the decision. The social indication should be clearly defined and the responsibility of the couple should be emphasized. A well defined and documented task for the clinical practice of prior physician and psychological discussion of tubal ligation is the basis of a present prospective study at the University Department for Women in Tubingen which is designed to show the positive psychological effects on the couple.


Multilocus behavior in random environments. I. Random Levene models. In this paper the consequences of natural selection acting on several loci simultaneously in a spatially fluctuating environment are described. The fitnesses of the genotypes are assumed to be additive both within and between loci. The environment is assumed to be made up of a very large (effectively infinite) number of patches in which fitnesses are assigned at random. The resulting deterministic model is called a Random Levene Model and its properties are approximate by a system of differential equations. The main equilibrium properites are that (1) the linkage disequilibrium is zero and (2) the correlations in fitness between alleles at different loci are the principle determinants of the dynamic inter-locus interactions. Although there is no epistasis as conventionally defined, the equilibrium state at the two loci are highly interdependent, the governing principle being that two alleles at different loci whose fitness are negatively correlated across environments have a higher overall fitness due to the reduction in their variance in fitness through the negative correlation. When a large number of loci are considered, they naturally fall into correlation groupings which lead to an enhanced likelihood for polymorphism over that predicted by single-locus theory.


On inverted repeat sequences in chromosomal DNA. It is suggested that chromosomal DNA should contain a class of palindromic reverse repeats, comparable in number to that of genes themselves, which are formed as follows:(1) a transcription-termination signal that follows the gene plus (on the complementary strand and located as near to the "anti-gene" as possible); (2) a second termination signal which actively prevents the accidental transcription of the anti-gene. Thus, the adjacent termination-anti-termination region of one strand would complement the anti-termination-termination region of the other.


Linkage analysis of the multilocus glucosephosphate isomerase isozyme system in sunfish (Centrarchidae, Teleostii). The purpose of the present investigation is to determine whether the two duplicated glucosephosphate isomerase (EC 5.3.1.9) loci Gpi-A and Gpi-B reside on the same chromosome in teleostean fishes. Interspecific sunfish hybrids were employed for the cross because of the different species-specific electrophoretic mobilities of the allelic isozymes at each GPI locus and because of their genomic compatibility. F1 sunfish hybrids, formed from a male warmouth (Lepomis gulosus) X female green sunfish (L. cyanellus) cross, were mated to form the F2 generation. The number of each of the nine different isozyme phenotypes, revealed by starch gel electrophoresis, was determined using 256 F2 individuals. The high frequency of recombinant phenotypes in the F2 generation indicated that the two GPI loci are not linked. An excess of F2 individuals heterozygous at both loci was observed and is interpreted as being caused by heterosis. The absence of linkage for the homologous loci encoding GPI subunits and for other multilocus isozyme systems is consistent with the postulate that the genomes of present-day vertebrates arose through one or more polyploidization events early in vertebrate evolution.


Polymorphism for dimerizing ability at the esterase-5 locus in Drosophila pseudoobscura. Three new alleles are reported at the esterase-5 locus of Drosophila pseudoobscura. All three of these alleles are different from those previously reported in their ability to dimerize. One allele will not form heterodimers or homodimers and exists only as a monomer. A second allele does form heterodimers but will not form homodimers. The third allele forms both hetero- and homodimers as well as forming monomers. Estimates of the frequency of these alleles in a natural population are given. The existence of these is discussed with respect to recently proposed models for a molecular mechanism for heterosis.


Two-way selection for growth rate in the common carp (Cyprinus carpio L.). The domesticated European carp was subjected to a two-way selection for growth rate. Five generations of mall selection for faster growth rate did not yield any response, but subsequent selection between groups (families) resulted in considerable progress while maintaining a large genetic variance. Selection for slow growth rate yielded relatively strong response for the first three generations. Random-bred control lines suffered from strong inbreeding depression and when two lines were crossed, the F1 showed a high degree of heterosis. Selection was performed on pond-raised fish, but growth rate was also tested in cages. A strong pond-cage genetic interaction was found. A theoretical explanation was suggested involving overdominance for fast growth rate and amplification through competition of intra-group but not inter-group variation.


Mapping of the chromosome of Mycobacterium phlei by means of mutagenesis of the replication point. The aim of the present work was to construct a replication map of the chromosome of Mycobacterium phlei. The method of mutagenesis of the replication point by means of nitrosoguanidine was applied to synchronously multiplying populations. Back mutations and forward mutations were induced in auxotrophic mutants PA met and PA leu as well as in double auxotrophic mutants with methionine as the reference marker and the following order of replication of eleven genes on the chromosome was thus established: leu-Eth, Res-Stm, Cyk-pur-met, arg, Cyk-Bac-inl.


Purification of xylanase from the wood-rotting fungus Trametes hirsuta. Xylanase (EC 3.2.1.8) was purified from a crude extracellular enzyme of the wood-rotting fungus Trametes hirsuta by a four-step procedure involving precipitation with ammonium sulphate, gel filtration on Bio-Gel P-10, column chromatography on DEAE-Sephadex A-50 and preparative electrophoresis on polyacrylamide gel. The isolated enzyme, electrophoretically homogeneous, was separated from beta-xylo-sylosidase and other hydrolytic enzymes studied. The analysis of the degradation products of water-soluble (4-0-methylglucurono)-D-xylan from willow by the purified xylanase showed it to be endoxylanase (beta-1,4-xylan xylanohydrolase).


Effect of some inhibitors on kanamycin formation by Streptomyces kanamyceticus. The formation of kanamycin is markedly inhibited by mercuric chloride, sodium iodoacetate, 2,4-dinitrophenol, sodium arsenite and sodium azide particularly when these are added at the start of fermentation. Less inhibition of kanamycin synthesis is observed in case of sodium 5,5-diethylbarbiturate, malonic acid, sodium arsenate and sodium fluoride. Inhibition of kanamycin synthesis is associated with growth inhibition in case of 2,4-dinitrophenol, sodium arsenite and sodium azide. Bacitracin and D-cycloserine have a stimulatory effect on kanamycin synthesis with slight inhibition of cellular growth. This stimulation might be due to accumulation of cell wallintermediates--aminosugar and sugar--which are shunted to the pathway of kanamycin synthesis. Penicillin lowers kanamycin synthesis by 65 percent as compared with 19 percent reduction of cellular growth. Chloramphenicol has a stimulatory effect at lower concentration (20 mug/ml), when it is added at 24 h of fermentation. At higher concentration (50 mug/ml) chloramphenicol shows marked inhibition of both cellular growth and antibiotic biosynthesis.


[A new pigmented antibiotic from a soil streptomycete]. An antibiotic  designated A28 was isolated from a soil streptomycete which showed wide antibacterial activity. The antibiotic was extracted and purified into a yellow powder. Its physico-chemical and antimicrobial properties indicated that it is a novel peptide antibiotic.


Bioengineering problems connected with the use of conventional and unconventional raw materials in fermentation. A review. Some bioengineering problems connected with the use of conventional and unconventional raw materials in fermentation research and industry are reviewed. They include the effect of the physical state of different substrates (solid, liquid, gaseous) and considerations of  physico-chemical processes, especially the identification of limiting steps. A new concept of classification of fermentors with respect to the macromixing properties is suggested and its applicability for different substrates is considered.


Effects of hypophysectomy and acute growth hormone treatment upon glucose metabolism in adipose tissues and isolated adipocytes of rats. Glucose oxidation and incorporation into lipid were measured in epididymal adipose tissues and isolated adipose cells of normal and hypophysectomized rats in an effort to determine whether the acute hypoglycemic effect of a systemic growth hormone (GH) injection was related to alterations in the glucose metabolism of adipose tissue. The rats were fed rat chow or a high sucrose diet and received 100 mug GH intraperitoneally 30 minutes or three and one-half hours before sacrifice. Hypophysectomized rats showed a lower plasma glucose as compared with normal rats on both diets. Thirty minutes after a GH injection there was a further decrease of the plasma glucose which, however, was not present in those rats receiving GH three and one-half hours before sacrifice. Adipose tissues from hypophysectomized rats fed the high sucrose diet showed a blunted insulin sensitivity as compared with normal rats on a similar diet. The insulin sensitivity of these tissues was further decreased 30 minutes after a GH injection. Basal glucose metabolism of isolated adipocytes from hypophysectomized rats, as compared with normal rats, was depressed if they were fed rat chow, was at normal levels if they were fed the high sucrose diet and was increased if they were fed the sucrose diet and received triiodothyronine and cortisone supplements. No manipulations of diet or hormonal treatments made the isolated adipocyte from hypophysectomized rats sensitive to insulin either 30 minutes or three and one-half hours after a GH injection. Since basal glucose utilization is not enhanced by GH injection and both the blunted insulin sensitivity of adipose tissue and the absent insulin sensitivity of adipopocytes would be expected to produce hyperglycemia rather than hypoglycemia, it is concluded that immediate systemic effects of a GH injection on carbohydrate metabolism are not related to changes in glucose metabolism of the peripheral adipose tissues.


Effects of 2-deoxy-D-glucose, oligomycin and theophylline on in vitro glycerol metabolism in rat adipose tissue: response to insulin and epinephrine. The effects of 2-deoxy-D-glucose (2DG), oligomycin and theophylline on the in vitro production and metabolism of glycerol and its response to insulin and epinephrine were studied in epididymal fat pads from fed rats. 2-DG failed to affect basal or epinephrine stimulated glycerol production but it decreased the uptake of 1-14 C-glycerol by the tissue and its conversion to glyceride-glycerol. Oligomycin also failed to affect the basal production of glycerol but it inhibited the effect of epinephrine on this parameter as well as the uptake and utilization of 1-14-C-glycerol. Theophylline enhanced the production of glycerol by the tissue and this effect was not further augmented by epinephrine. Theophyline also inhibited the uptake and utilization of 1-14C-glycerol; the most pronounced effect of theophylline was observed in the formation of 14C-fatty acids from 1-14C-glycerol in the presence of glucose. Insulin, but not epinephrine, decreased the inhibitory effect of theophylline on glycerol utilization. It is concluded that these compounds affect more intensely the ability of adipose tissue to metabolize glycerol than to release it through lipolysis. The pathway for glycerol utilization in adipose tissue appears to be more sensitive to changes in the availability of ATP than the mechanisms responsible for the release of glycerol from the tissue.


Theophylline stimulation of gluconeogenesis from alanine in normal and sympathectomized rats. The influence of theophylline ethylenediamine (100 mg/kg i.p.) on gluconeogenesis was studied in normal and in adrenodemedullated and reserpinized rats after overnight fasting by measuring the time-course of Alanine-14C incorporation into Glucose-14C. In the normal rat, theophylline produced a moderate hyperglycemia associated with an increased conversion of alanine to glucose at all time intervals. In addition, a marked rise of plasma levels of insulin and glucagon was observed. In sympathetctomized rats, plasma glucose and gluconeogenesis were again enhanced by theophylline, but the pattern of these modifications differed from that of normal rats since the peak values occurred earlier. Subsequently, both parameters rapidly declined reaching values lower than controls at the end of the experiment. Insulin response to theophylline was higher in sympathectomized animals in comparison to normal rats, while glucagon response was approximately of the same magnitude in the two groups. From these findings it was concluded that theophylline is able to stimulate gluconeogenesis from alanine both in the normal and sympathectomized rat. The different pattern of alanine conversion to glucose seems to depend on the different participation of insulin and catecholamines in the two groups.


Pituitary-thyroid axis reaction after myocardial infarction. Within the first 36 hours following myocardial infarction, serum total thyroxine (T4) levels were supranormal in most cases in contrast to normal thyrotropin values. After one week, T4 levels dropped to normal while TSH values rose significantly. These findings suggest that, in the acute phase of myocardial infarction, the secretion of thyroid hormones is increased, thereby inhibiting the pituitary thyrotropes. The stimulation of thyroid secretion might be due to the high levels of blood catecholamines generally found in patients with myocardial infarction.


Protein metabolism in hypo- and hyperstimulated rat thyroid glands. II. Degradation of newly formed thyroidal proteins. The rate of degradation of total thyroidal proteins is modified in differently stimulated glands. It is slowed down in hypostimulated thyroids and accelerated in hyperstimulated ones. Comparative evaluation of the rates of degradation (either in absolute terms - DPM/mg of tissue or as specific activity) of different proteins shows that a modified hormonal state affects the degradation of the thyroglobulin much more significantly than the degradation of non-thyroglobulin proteins. In the absence of thyrotropic hormone (TSH) the degradation of thyroglobulin is almost completely inhibited, while with excess of hormone it is dramatically accelerated. Comparing the TSH action on the synthesis with its effect on the degradation of thyroglobulin, it appears that it has a much stronger effect on the process of degradation than on the process of synthesis. This means that TSH significantly modifies the turnover of thyroglobulin. This effect of TSH leads, in chronically hypo- or hyperstimulated glands to the new levels of colloidal thyroglobulin which are highly increased in hypostimulated and significantly decreased in hyperstimulated glands. These results are in perfect agreement with the classical morphological description of hypo- and hyperstimulated glands.


Effect of amino acid reutilization in the determination of protein turnover in mice. Using different decay data of "in vivo" pulse-labeled protein, the half-life values of different fractions from mouse liver, heart, striated leg muscle, jejunum and epidydimal fat pads were calculated by considering labeled intracellular amino acid reutilization. This was accomplished by substracting the pool amino acid decay rates from the protein decay rates. The extent of reutilization of these labeled amino acids is considerable as shown by the differences found between the corrected and uncorrected half-life calculations. The half-life values obtained for total protein in liver, striated muscle, heart, duodenum and epidydimal fat pad were: 0.57, 1.81, 2.13, 0.68 and 0.42 days, respectively.


[Synthesis of some aminoacyl-AH-sepharoses via the N-carboxyanhydride method (author's transl)]. (Aminoacyl)n=1-10-AH-Sepharoses can easily be prepared by the N-carboxy anhydride method from amino acid N-carboxy anhydrides and AH-Sepharose 4B at pH 10.2, 0degreesC in aqueous borate buffer. Syntheses of cystinyl-, cysteinyl-, isoleucyl-, leucyl- and phenylalanyl-AH-Sepharoses are described. An improved preparation for cystine bis(N-carboxy anhydride) is presented.


Effects of acrosin inhibitors on the soluble and membrane-bound forms of ram acrosin, and a reappraisal of the role of the enzyme in fertilization. When denuded ram spermatozoa were suspended in weakly buffered 0.25M sucrose, the acrosin remained bound to the acrosomal membranes of the sperm heads. Media containing CaCl2 caused complete solubilization of the enzyme. Effects of acrosin inhibitors on soluble and bound enzyme were studied in Tris HCl(pH 8.2) containing sucrose. Denuded spermatozoa were used as a preparation of bound acrosin. Trasylol (Kunitz basic pancreatic trypsin inhibitor) acted more strongly on bound scrosin than on soluble acrosin, but soya-bean trypsin inhibitor acted more strongly on soluble acrosin. At concentrations 0.5 - 2.0muM, the inhibitors isolated from ram acrosomes and from ram seminal plasma inhibited soluble acrosin but had negligible effects on bound acrosin. However, bound acrosin was sensitive to high concentrations of the acrosomal inhibitor. The two forms of acrosin were inhibited to about the same degree by p-aminobenzamidine and also by Tos-Lys-CH2Cl. It is proposed that membrane-bound acrosin is the form that functions in penetration of the zona pellucida, and that a role for acrosin inhibitors is suppression of an antifertility effect of soluble acrosin on mammalian eggs. This hypothesis is supported by 1) the results of work on the impaired fertilizing capacity of rabbit spermatozoa that have been treated with acrosin inhibitors, 2) the anti-fertility effects on hamster eggs of solutions of acrosin and of bovine trypsin, and 3) the results in this paper.


Chemical syntheses of novel fluorescent-labelled fatty acids, phosphatidylcholines and cholesterol esters. The synthesis of a novel class of fluorescent-labelled fatty acids of different chain lengths and unsaturation, phospholipids and cholesterol esters has been developed. The following omega-anthracene-labelled cis-unsaturated fatty acids have been synthesized: omega-(9-anthryl)-6c-octenoic, -7c-nonenoic, -10c-dodecenoic, -6c,9c-undecadienoic, -10c,13c-pentadecadienoic acid. They have been introduced into the 2-position of 1-stearoyl- and 1-linoleoyl-3-sn-glycerophosphocholine and cholesterol. Mass spectroscopy, 1H-NMR, IR and fluorescence spectroscopy and different chromatographic procedures have been applied to confirm and characterize their structures. The properties of the different fluorescent-labelled phosphatidylcholines in monomolecular films have been determined by the Langmuir technique.


Pryogalloloestrogens -- a new group of oestrogen metabolites. After incubation of radioactive catecholoestrogen monomethyl ethers with rat liver slices the following well known metabolic pathways were observed: 1) demethylation, 2) 16alpha-hydroxylation, 3) oxidoreduction at C-atom 17, and 4) conjugation with glutathione, sulphuric acid and glucuronic acid. In addition, for the first time a further aromatic ortho-hydroxylation, leading to pyrogalloloestrogen derivatives, was detected. Thus, the incubation of 2-methoxyoestrone yielded 2,4-dihydroxyoestrone 2-methyl ether as the main metabolite of the lipophile fraction. Under the same conditions, 4-methoxyoestrone was converted to 2,4-dihydroxyoestrone 4-methyl ether and 2,4-dihydroxyoestradiol-17beta 4-methyl ether; these compounds were the quantitatively most important metabolites not only in the lipophile but also in the sulphate and glucuronide fractions. The identity of these new metabolic products was established by chromatography, microchemical reactions and recrystallisation to constant specific radioactivity.


Pyrogalloloestrogens: enzymic methylation of pyrogalloloestrogens and interaction of pyrogalloloestrogens with the enzymic methylation of catecholamines in rat liver in vitro. During incubation of 2,4-dihydroxyoestrone with the 105000 X g supernatant of rat liver in the presence of S-adenosyl-[Me-14C]methionine, the formation of radioactive mono- as well as dimethyl ether derivatives was demonstrated. The products were identified as: 2,4-dihydroxyoestrone 2-methyl ether, 2,4-dihydroxyoestrone 3-methyl ether, 2,4-dihydroxyoestrone 4-methyl ether, 2,4-dihydroxyoestrone 2,3-dimethyl ether, 2,4-dihydroxyoestrone 2,4-dimethyl ether and 2,4-dihydroxyoestrone 3,4-dimethyl ether. The monomethyl ethers were the main products; within this group the 3-methyl ether of 2,4-dihydroxyoestrone was the main metabolite. Among the dimethyl ether derivatives, the 2,4-dihydroxyoestrone 2,3-dimethyl ether represented the quantitatively most important product. When 2,4-dihydroxyoestrone 2-methyl ether was incubated under the same conditions, 2,4-dihydroxyoestrone 2,3- as well as 2,4-dimethyl ether was formed. The 2,3-dimethyl ether was again the main metabolite. The incubation of 2,4-dihydroxyoestrone 4-methyl ether yielded the 2,4- and 3,4-dimethyl ethers, the first being the main product. In contrast, the 3-methyl ether of 2,4-dihydroxyoestrone was not further methylated by the catechol methyltransferase preparation. In further experiments, the effect of the pyrogalloloestrogen and its monomethyl ether derivatives on the enzymatic methylation of catecholamines was investigated. It was demonstrated that the methylation of adrenalin and dopamine was competitively inhibited by 2,4-dihydroxyoestrone and the 2,4-dihydroxyoestrone monomethyl ethers. Only a weak inhibitory effect was observed with the 3- and 4-monomenthyl ethers (Ki values 200 and 160muM). The unsubstituted pyrogalloloestrogen produced a marked inhibition (Ki value 50muM), but the strongest inhibition was found with the 2-monomethyl ether of 2,4-dihydroxyoestrone (Ki value 14muM). The extent of inhibition caused by the addition of the 2-monomethyl ether of 2,4-dihydroxyoestrone was thereby in the same range as the inhibition caused by pyrogallol and the catecholoestrogens.


[Isolation of an amatoxin binding protein (ABP) different from RNA-polymerase B and C ((author's transl))]. During the isolation of the amatoxin RNA-polymerase B-complex from calf thymus tissue we also isolated a protein (ABP) which shows such strong affinity to [3H)amanin that significant binding occurs at low concentrations (10-7M) of the label. The presence of a new amatoxin-complex is demonstrated by coprecipitation of amatoxin and ABP with ammonium sulphate and the common chromatography on phosphocellulose and Sephadex G-25. The new protein ABP is characterized by denaturating sodium dodecylsulphate-gel electrophoresis. The molecular masses of both main bands - possibly subunits of ABP - are determined as 100000 and 10000 - 15000 Dalton and different from the subunit pattern of RNA-polymerases B and C.


[Sexually differentiated metabolism of testosterone in liver slices of mouse, and its alteration by a mutation in the X-chromosome that results in testicular feminization]. 1. Sexual differentiation of the metabolism of testosterone in liver slices of normally developed, sexually mature mice: Sexual differentiation in the mouse, unlike that in the rat, shows a high degree of uniformity: Where the formation of metabolites with the composition C19O2 is markedly greater in one sex, then this is invariably the male. The formation of C19O3 steroids and 4-androstene-3,17-dione, and the turnover of testosterone show no marked sexual differences, although the sum of the C19O2-type delta4-hydrogenation products of testosterone is significantly greater in the male. This apparent discrepancy is explained by the fact that the sum of the delta4-hydrogenation products represents no more than 10% of testosterone turnover. Thus, sexual differences in the formation of individual delta4-hydrogenation products are not apparent from a consideration of the overall turnover of testosterone. 2. Sexual differentiation of testosterone metabolism studied in genetically male litter mates, carrying the X-chromosome-bound mutation and showing testicular feminization (Tfm): The Tfm mutation (genotype XTfm Blo/Y; Blo = coat colour gene Blotchy) results in a feminization of testosterone metabolism. Where the level of testosterone metabolites is significantly higher in the normal male than in the normal female, the Tfm mutation shows a level that is significantly lower than in the normal male, and which, in most cases, is the same as that in the normal female. The concentration of three metabolites (3alpha- and 3beta-hydroxy-5beta-androstan-17-one, and 5beta-androstane-3,17-dione), which do not show sex-based differences, were significantly increased in the Tfm mutation. The Tfm mutation therefore effects the formation of all ring A hydrogenation products of type C19O2 (with the single exception of 5bets-androstane-3alpha,17beta-diol). It does more than simply equalize sexual differences by feminization. It has no effect on the hydroxylation of testosterone, or on its 17beta-dehydrogenation to 4-androstene-3,17-dione. The consequences of the Tfm mutation for the liver are irreversible: The formation of 5alpha-androstane-3,17-dione, which is a representative parameter for the sexual differentiation of testosterone metabolism, is not influenced by the injection of testosterone (15 mg i.p. 6 days before investigation).


Paracentric inversion of a human chromosome 7. In the course of chromosome studies of atomic bomb survivors in Hiroshima using the trypsin-G-banding and Q-banding methods, a 40-year-old male was found to have an abnormal banding pattern in the long arm of a chromosome 7, although no such abnormality was detected by ordinary staining method. Since all other chromosomes apparently had normal banding patterns, the abnormality was determined to be a paracentric inversion of a chromosome 7, which is described as 46, XY, inv (7) (q22q31). This is the first demonstration of a possible paracentric inversion in man.


On the population genetics of phenylketonuria in the G.D.R. By means of the analysis of the geographical distribution of the birth-places of cases with phenylketonuria, who were detected in a nation-wide screening for phenylketonuria by the Guthrie test during a period of more than 5 years, 3 areas with a remarkably different frequency can be recognized: a) in the districts of Cottbus, Leipzig, Halle and Frankfort the frequency of the phenylketonuria is amounting to 1:6275, of the phenylketonuria-gene to 0.0126 and of the heterozygotes to 2.49%; b) in the remaining districts with the exception of two districts the frequency is significantly lower (1:11272, 0.0094, 1.86%); c) in the districts of Karl-Marx-Stadt and Neubrandenburg this metabolic disease is relatively rare (1:14769, 0.0082, 1.63%). In regions with a high frequency of the phenylketonuria the permanent mild hyperphenylalaninaemia is also twice more frequent than in the remaining country.


DNA replication patterns of the early S phase from amniotic fluid cells as revealed by a Giemsa staining technique. We report on DNA replication patterns of the early S phase demonstrated by a BUdR/Hoechst (33258)-fluorescence/Giemsa technique. Replication starts in R bands and spreads within 1 hr over the whole chromosome complement with exceptions concerning heterochromatin. The beginning and the duration of replication are discussed.


Centric fission of chromosome no. 4 in the mother of two patients with trisomy 4p. Centric fission of chromosome No. 4 was found in the healthy mother of two children with trisomy 4p. The two telocentrics derived are stable and show no evidence of fusing again. The 2:3 ratio of unbalanced offsprings born to the proposita indicates that, contrary to the evidence emerging from studies in other species, the risk for the production of unbalanced gametes is high.


5-methylcytosine in heterochromatic regions of chromosomes in Bovidae. The centromeric regions of cattle, goat and sheep chromosomes bind anti-5-MeC as revealed by immunofluorescence technique, indicating concentration of 5-MeC at these heterochromatic regions. The centromere of the submetacentric X of cattle remains nearly unstained and so do the centromeres of the acrocentric X chromosomes in goat and sheep. The short arm of the cattle Y exhibits strong anti-5-MeC binding whereas the tiny Y chromosomes of goat and sheep contain no brightly fluorescent material.


Pyruvate kinase isozymes in man. I. M type isozymes in adult and foetal tissues, electrofocusing and immunological studies. Anti human M2 type and anti human L type pyruvate kinase sera allowed us to distinguish two groups of pyruvate kinase in man. Erythrocyte and liver (L type) enzymes on the one hand were inhibited by anti L and not all by anti M2 serum; pyruvate kinase from all the other tissues on the other hand were inhibited by anti M2 and not at all by anti L serum. This latter group represent the M type pyruvate kinase isozymes. The M type isozymes have been studied by electrofocusing in thin layer acrylamide-ampholine gel. In adult tissues 4 types of isozymes were found, designated, from acid to alkaline pH, as M2 (predominant form in spleen, leukocytes, lung...), M3, M4 and M1 (predominant form in muscle and brain). In foetal tissues an extra band M2, called M2f, more anodic than M2, was added to the previously described isozymes. Except in brain (in which the isozymes M2, M3, M4 and M1 were found), the most anodic bands (M2f, M2 and M3) were predominant in all the foetal tissues. The isozymes M2f and M2 seem therefore to be the original M type pyruvate kinase forms from which the other isozymes issue. The rate of each isozyme seems to depend on tissue factors characterizing the state of differentiation of some tissues, as indicated by the ability of adult muscle extracts to change the isozymes M2 and M3 into more cathodic forms.


Cytogenetic investigations in a new case of Bloom's syndrome. Cytogenetic studies of an 8-year-old caucasian girl with typical but mild manifestation of Bloom's syndrome showed a characteristic increase of homologous chromatid translocations and prematurely condensed chromosomes. The average frequency of sister chromatid exchanges (SCE) in lymphocytes with 133 was much higher than in skin fibroblasts with 49. The inter- and intrachromosomal distributions of SCE in lymphocytes were analyzed.


Possible genetic consequences of relaxed selection against common disorders with complex inheritance. With increasing success in medical treatment for many common disorders with complex inheritance, it is expected that the incidence of patients requiring treatment will gradually rise from generation to generation. While this is theoretically true for a long range of future, the pattern of possible increase in the frequency of a disorder resulting from complete cure has been examined with special reference to the next few generations. It was shown that the maximum possible increment will take place in the first generation, with additional increment rapidly diminishing with subsequent generations. For congenital heart disease, for example, the restoration of full fertility to all of the patients would incur an increase in the frequency at birth of the disease by about 10% after 5 to 10 generations.


[A case of testicular feminization with the karyotype 47, XXY (author's transl)]. A case of testicular feminization is described, who has the karyotype 47, XXY in all analysed metaphases from blood and skin cultures. The question is discussed whether the karyotype 47, XXY is compatible with the syndrome of testicular feminization and which conditions are required to allow the hitherto known transmission of testicular feminization in presence of the gonosomes XXY. A review of literature is given.


Is haemoglobin G alpha Philadelphia linked to alpha-thalassaemia? The question, "Is Hb G alpha Philadelphia linked to alpha-thalassaemia?" was first posed because the abnormal haemoglobin is found in heterozygotes at a concentration greater than 25%, the proportion predicted from a 4 alpha-chain gene model. Globin chain biosynthesis was studied in a West Indian family in which one parent had beta + thalassaemia and the other was heterozygous for the G alpha Philadelphia chain gene. The former had a globin chain production ratio alpha/beta well above 1, while the latter had a ratio significantly less than 1. One child of the marriage had inherited the beta + thalassaemia from one parent and the G alpha Philadelphia chain gene from the other and showed the typical picture of alpha/beta-thalassaemia (alpha/beta ratio slightly above normal). It is explained in the discussion that the evidence favours a close linkage of 2 alpha-chain genes.


Four familial translocations ascertained through spontaneous abortions. In a study of 514 spontaneous abortions, 194 were found to have a chromosome anomaly. Of these, 4 (2.1%) were unbalanced translocations. Three of the translocations were Robertsonian (13q14q) and one was reciprocal. Each translocation was ascertained independently and each was associated with a balanced rearrangement in a carrier parent.


Aspermia, associated with a presumably balanced X/autosomal translocation karyotype 46, Y, t (X;5) (q28;q11). A new case of X/autosome translocation in a male patient is described. Azoospermia and Klinefelter like stigmata can be explained as a consequence of the balanced translocation, or by disturbed X-chromosomal inactivation during spermiogenesis.


Nonrandom distribution of chromosome breaks in cultured lymphocytes of normal subjects. Breakpoint distribution was studied from cultured lymphocytes on 7653 metaphases from 524 subjects whose karyotypes were normal. The mean break rate was 5% in both sexes. The frequency increased significantly after 40 years and varied during the year. The location of the breaks was very different from the expected random distribution. The break frequency for each chromosome was different according to the type of break (chromatid, simple chromosomal and chromosomal involving rearrangements). The location of the breaks was also studied according to type of band and with respect to the centromere. A comparison between spontaneous breaks, X-ray induced breaks, breaks in Fanconi's anemia had in congenital rearrangements, show very significant differences.


A cytogenetic study of human spontaneous abortions using banding techniques. The karyotypes of 941 singleton and 42 twin abortuses and 4 cystic placentae were determined. 30.5% of the singletons were chromosomally abnormal; 49.8% of these were trisomic, 23.7% X-monosomics and 17.4% polyploid. 143 trisomies were identified by banding; over a third had an extra chromosome 16, more than 10% an extra 21 or 22 and about 5% an extra 2, 18 or 15. Examples of trisomy 3, 4, 8, 9, 10, 13, 14 and 20 were also encountered. Using the data from two other published studies, the prevalence of different trisomies was estimated and an attempt was made to relate the karyotype of the conceptus to its subsequent development.


[Dermatoglyphics of homo- and heterozygotes for Wilson's disease (hepatolenticular degeneration) (author's transl)]. Dermatoglyphics of 11 patients with Wilson's disease and 16 of their clinically asymptomatic relatives of first degree were investigated; 11 of the latter ones were heterozygous in agreement with the turn over rates of Cu-67, 12 under the assumption of autosomal recessive inheritance. On the finger tips the Mb. Wilson patients showed 52.7% whorls, their heterozygous relatives about 40%; compared with our controls (males 33.16%, females 28.82%, Aue-Hauser, 1970) that means a strong increase of this pattern type. On the palm the high frequency of hypothenar patterns in homo- and heterozygotes for Wilson's disease and of loops with accessory triradius in the 4th interdigitum of the patients with Wilson's disease was striking.


Partial trisomy 11,46,XX,-3,-20, + der3, + der20,t(3:11:20), resulting from a complex maternal rearrangement of chromosomes 3, 11, 20. Clinical findings of partial trisomy 11p are described in a patient bearing t(3;11;20) (p13;p11;q13). The translocation was present in balanced form in her mother (46, XX)t(3;11;20) (p13;p11;q13).


A boy with 47,X,del(X)(p11leads to q13:q21leads to q24),del(Y)(q11):reexamination of a case previously described as 47,XX,?Yq-. Before the introduction of the banding technique we studied a boy presented as a case of 47, XX,?Yq-(Christensen and Nielsen, 1971). The case has been restudied with the use of different types of banding, which revealed the following karyotype 47, X,del(X)(p11 leads to q13::q21leads toq24), del(Y)(q11).


Possible linkage of HL-A and GLO.

Possible linkage of HL-A and GLO. In 21 informative families with 60 children, a possible linkage between HL-A and GLO was found (recombination fraction approximatively 0.15). The sequence of the loci on chromosome 6 might be GLO, HL-A, PGM3, MNSs.


A 22/22 translocation carrier with recurrent abortions demonstrated by a Giemsa banding technique,. A 22/22 Robertsonian translocation has been identified in a woman with recurrent abortions by a Giemsa banding technique. Cytogenetic studies of the embryonic tissue derived from one of her spontaneous abortions have demonstrated that the aborted fetus had a 46,XX,-22,+t(22q22q) karyotype.


Primary culture and maintenance of steroid-secreting human placenatal monolayers. A simple method is described for primary culture and for maintenance of hormone-producing cells from normal human placenta. A consistent yield of cells was obtained and an average survival of 3 to 4 months in culture using 1 mm3 explants from the most vascular area of the placentas. These explants were placed in a variety of culture media in 30 ml flasks and incubated at 37 degrees C in an atmosphere of 5% CO2 and 95% air. The best yields in terms of cell growth were observed with Eagle's MEM (minimum essential medium) with supplements of horse serum and fetal calf serum or human cord serum. (Ham's F-10 with supplement of horse serum and fetal calf serum supports growth for the longest period and media containing human cord serum had the best yield of steroids.


The different roles of serum and calcium in the control of proliferation of BALB/c 3T3 mouse cells. Proliferatively inactive BALB/c 3T3 mouse cells in dense cultures initiate a growth-division cycle upon exposure to fresh calf serum in a low-calcium (0.01 mM) medium. If these calcium-deprived cells are not supplied with calcium sometime during the first 10 hours after serum stimulation, they will rapidly return to a proliferatively inactive state without initiating DNA synthesis. The prereplicative development of such stimulated calcium-deprived cells appears to stop at an advanced stage, because addition of calcium as late as 10 hours after serum exposure rapidly initiates DNA synthesis, and enables the culture's DNA-synthetic activity subsequently to reach its peak value at the same time as in control cultures.


Reduced toxicity of amphotericin B methyl ester (AME) vs. amphotericin B and fungizone in tissue culture. The comparative toxicities of amphotericin B methyl ester (AME), the parent antibiotic amphotericin B (AB), and the deoxycholate solubilized complex of AB, Fungizone (FZ), toward five cell lines has been determined as measured by early membrane damage (51Cr release), 24 hr survival, 72 hr viability, and growth rate. Cells used were of turtle (TH-1), marsupial (PT K2), human MA 160), rabbit (RK-13) and hamster (BHK-21) origin. AME: (a) caused less membrane damage at 1 hr than AB or FZ; (b) was less toxic than AB or FZ as indicated by 24 hr cell survival and 72 hr cell viability; and (c) was required in higher levels than AB or FZ to reduce the growth rate of all five cell lines. Spectrophotometric analysis of residual polyene levels indicated that AME had good stability in tissue culture medium. Previous studies have indicated that AME has the same in vitro antifungal activity as the parent antibiotic AB (1, 2). These findings suggest that AME may prove to be superior to AB and FZ for use as an antifungal agent in tissue culture systems.


Ultrastructure and surface topography of aggregates of human limb mesenchymal cells (HLM15) in vitro. Tissue-like aggregates of human embryo fibroblasts can be created in vitro by limited aspiration of cells released from substrate during subcultivation. Aggregates increase in size, exhibit intercellular junctions, display a surface topography characteristic of cellular movement, elaborate an extracellular matrix and possess features of cellular death and phagocytosis. These cells, when introduced to a new culture environment, do not migrate away from one another as is common when a primary culture is started from tissue fragments. Instead, cells exhibit continued contact with each other, and develop complex junctional structures during that association. Cellular aggregates generated in this manner may provide a useful system for providing further information on cellular adhesion, intercellular communication, morphogenetic cell movements and the mechanisms of cell death.


Viral contamination of a mosquito cell line, Aedes albopictus, associated with syncytium formation. Viral contamination associated with syncytium formation in two sbulines of Singh's Aedes albopictus cell cultures was investigated. Electron microscopy of the syncytia revealed the presence of five different types of virus-like particles, which morphologically resembled the parvo-, picorna-, toga-, and orbi-, and bacterial viruses. When a virus-free subline of the A. albopictus cells (SL3) was inoculated with extracts of the syncytium-forming A. albopictus cells, the parvo-, toga-, and orbi-type viral agents were consistently observed. Among these three agents, the togavirus-type agent is most likely responsible for the syncytium induction. Serological examination of the infected cell extract indicated that at least one of three virus-like agents, presumably the togavirus-type agent, was related to Chikungunya. O'nyong-nyong, and Western equine encephalomyelitis viruses (alphaviruses of the Togaviridae), but separable from these.


Growth of human muscle in tissue culture. An improved technique. A method is described for the culture of normal and diseased human muscle cells. Cell outgrowth was obtained from 63/63 biopsies, and cells differentiated to form myotubes in 57/63 biopsies. The culture technique used readily permitted the growth of both normal and diseased human muscle cells.


Antibody-dependent cell-mediated cytotoxicity in the rat. The role of macrophages. Experiments are described in which the inactivation of macrophage activity by quartz particles is used to investigate the role of macrophages in antibody-dependent cell-mediated cytotoxicity in the rat. The results confirm previous findings which indicated that macrophages play no significant role in assays using cell line cells as targets. On the other hand, previous suggestions that macrophages are active against antibody-coated chick erythrocytes cannot be substantiated. In fact, it is shown that macrophages can play a protective role, and that inhibiting macrophage activity in peritoneal exudates leads to a spectacular increase in antibody-dependent lysis of chick erythrocytes. It is suggested that the confusion surrounding the role of macrophages in this assay has resulted from the failure to recognize that adherence techniques such as carbonyl-iron treatment of cell suspensions can result in substantial depletion of non-phagocytic adherent cells.


Post-operative depression of antibody-dependent lymphocyte cytotoxicity following minor surgery and anaesthesia. Leucocytes taken 1 day post-operatively from patients who had undergone surgery under general anaesthesia for benign breast disease showed a significantly diminished capacity to induce lysis of antibody-coated target cells compared with those taken pre-operatively from the same patients. No significant fall in PHA responsiveness was observed in these leucocytes in the post-operative period. This indicates a high sensitivity of the cell types involved in the antibody-dependent cell-mediated cytotoxicity reaction to the suppressive effect of surgery and anaesthesia. Plasmas taken post-operatively from these patients were effective in diminishing the capacity of leucocytes from healthy untreated donors to initiate antibody-coated target cell lysis compared with pre-operative plasmas although the plasma cortisol levels in these samples did not differ significantly. Possible mediators of this suppressive effect and its significance are discussed.


Spontaneous rosette formation by mouse L cells. Mouse fibroblasts form spontaneous rosettes with sheep erythrocytes and with red cells from several other species. They do not form C3 or Fc rosettes, and they do not bind small amounts of aggregated gammaglobulin. Spontaneous rosette formation is maximum at 37 degress and minimum at 4 degrees. The reaction is complete in 30 min. Antisera to fibroblast membrane components do not inhibit rosette formation. Rosetting is inhibited by fixation of the cells with formaldehyde or glutaraldehyde as well as by killing with phenol or by prolonged incubation with sodium azide. The rosette reaction is inhibited by vinblastine but not by cytochalasin B. From these data on the kinetics, temperature and drug sensitivities it is suggested that T cells and fibroblasts form spontaneous rosettes by different mechanisms.


Use of (75Se)L-Selenomethionine as a label for lymphoid cells. [75Se]L-selenomethionine can be employed as a label for following lymphocytes in vivo. Its properties are broadly similar to those of 51Cr with regard to uptake, release of the isotope on cell death and reutilization; thus relatively high levels of 75Se in lymphoid tissue indicate the presence of viable labelled cells. It differs from 51Cr in that active release of selenium occurs from the labelled cells when the ambient cell concentration is low. No significant reutilization of leaked label occurs. Leakage of 75Se label occurs in vivo; this differs in the spleen and lymph nodes. Evidence is presented to suggest that 75Se losses reflect the transit times of cells through lymphoid organs.


Heterogeneity among preparations of crude trypsin used to disaggregate the human tonsil. Crude trypsin is the agent of choice for the  disaggregation of human tonsil. A great variability which is not related to specific activity exists among lots of crude trypsin. An efficient lot of crude trypsin is necessary to obtain optimal proportions of tonsillar cells for the purification of lymphocytes and plasma cells by a previously reported technique.


Suppression of the primary immune response to keyhole limpet haemocyanin by antimacrophage globulin. Thioglycollate-stimulated, adherence and density gradient-enriched peritoneal exudate cells were used for the preparation of a specific, highly active antimacrophage reagent. After absorption with mouse lymphoid cells, an antimacrophage globulin (AMG) fraction was shown to be cytotoxic for peritoneal exudate macrophages but not spleen, lymph node or thymus cells. The AMG suppressed both the in vivo primary serum antibody and spleen PFC responses to KLH.


Detection of circulating soluble immune complexes in patients with various renal diseases. Sera from patients with various types of glomerulonephritis (GN) as well as sera from rabbits with acute serum sickness were studied for the presence of circulating immune complexes (IC). The method used is based on the observation that IC inhibit the uptake of IgG aggregates by guinea-pig peritoneal macrophages. Inhibition significantly greater than with normal human sera was found with sera of patients with membranous GN, membranoproliferative GN, focal glomerular sclerosis, minimal change nephrotic syndrome, acute septicaemic glomerular diseases and systemic lupus erythematous. IC were also detected in rabbits with acute serum sickness during the period  of immune elimination.


Autoimmune murine thyroiditis. VIII. Role of different thyroid antigens in the induction of experimental autoimmune thyroiditis. Mice of the C57Br strain, which are susceptible to the induction of autoimmune thyroiditis with mouse thyroglobulin, and C57Bl mice, which are resistant, were immunized with human and rabbit thyroglobulins in Freund's complete adjuvant. Susceptible strain C57Br developed higher degrees of thyroid infiltration than the resistant strain. The results indicate that the responses to xenogeneic (foreign) thyroglobulins parallel allogeneic and syngeneic (mouse) thyroglobulin. BSVS mice, which are highly susceptible to thyroiditis, were immunized with mouse thyroid extract from five different mouse strains including syngeneic antigen. Recipients of C57Bl and DBA thyroid extracts showed lower indices of pathology than recipients of similar extracts from C3H, BSVS and non-inbred CF-1 mice. The results suggest that there is a difference in the immunogenicity of mouse thyroid extracts from different strains. Purified thyroglobulin was prepared from congenic strains B10.D2 (H-2d, resistant) and B10Br (H-2k, susceptible). H-2k thyroglobulin gave a greater response in both H-2k and H-2d mice than H-2d thyroglobulin.


The immune mechanism which expels the intestinal stage of Trichinella spiralis from rats. The immunological response of rats to the intestinal phase of Trichinella spiralis was assessed using criteria derived from pervious studies with the nematode Nippostrongylus brasiliensis in rats and mice. In adult rats, the duration of infection with either parasite is similar and both infections are prolonged in young and lactating rats. As previously shown with N. brasiliensis, immunity to T. spiralis was transferred to recipients with antiserum or mesenteric lymph node cells from immune donors and antisera and cells given in combination had an additive effect. Signs of damage similar to that caused by antibodies in N. brasiliensis appeared in T. spiralis adult worms as the infection progressed and this damage occurred earlier in animals given antiserum or cells. On the basis of these results, it is concluded that the immunological control of adult T. spiralis requires both antibodies and cells, but the relative importance of these components and the way in which they affect T. spiralis requires further analysis.


Tissue inactivation of slow reacting substance of anaphylaxis. The release of slow reacting substance of anaphylaxis (SRS-A) from sensitized guinea-pig lung challenged with antigen was followed by a fall in the activity of this mediator in the diffusate when the incubations were continued for 3 h. The inactivating principle was also present in normal lung in addition to other normal guinea-pig tissues such as the liver, kidney, spleen, ileum and skin. No acitivity was found in muscle. Evidence is provided that some of this SRS-A-inactivating activity was due to tissue arylsulphatases. These were measured by hydrolysis of p-nitrophenyl sulphate (p-NPS) and p-nitrocatechol sulphate (p-NCS), which reflect activities of arylsulphatase II A and II B respectively. Hydrolytic activity for p-NCS was present in all tissues with SRS-A-inactivating properties, whereas only lung, liver and skin tissue hydrolysed p-NCS and p-NPS. Following passage of a cell-free lung homogenate through a column of Sephadex G-200 the p-NCS hydrolysing and SRS-Z-inactivating activities eluted together, with molecules having a molecular size of approximately 150,000 Daltons, p-NPS hydrolysing activity being destroyed during the preparation of the homogenate. The release of arylsulphatase from sensitized tissue was not dependent on the presence of specific antigen. These experiments suggest that inactivation of SRS-A is related to tissue arylsulphatase and that these enzymes may play a role in the expression of the effective levels of this mediator within the tissues.


Effect of fatty acids on Staphylococcus aureus delta-toxin hemolytic activity. The lysis of human erythrocytes by Staphylococcus aureus delta-toxin proceeded without a lag and was directly proportional to toxin concentration and temperature of incubation. Lysis was complete within 8 min. Addition of saturated, straight-chain fatty acids of 13 to 19 carbons increased the activity of delta-toxin, whereas those with 21 to 23 carbons were inhibitory. Palmitic acid was the fatty acid most active in augmenting delta-toxin, but its effect could be abolished by the simultaneous addition of either tricosanoic acid or egg lecithin.


Identification of lethal toxin with the thermostable direct hemolysin produced by Vibrio parahaemolyticus, and some physicochemical properties of the purified toxin. Lethal toxin was purified extensively from the culture filtrate of a Kanagawa phenomenon-positive strain of Vibrio parahaemolyticus. The purified toxin was a protein, and its homogeneity was demonstrated by sodium dodecyl sulfate-polyacrylamide gel disc electrophoresis and analytical ultracentrifugation. It was demonstrated that the thermostable direct hemolysin was identical to the lethal toxin and that it was the main, if not only, lethal toxin in the culture filtrate. The purified toxin had a lethal effect when injected into mice either intravenously or intraperitoneally. Its lethal effect was very rapid, a dose of 5 mug of toxin per mouse killing the animals within 1 min. The lethal activity was inhibited by a ganglioside mixture. Some physicochemical properties of the purified toxin are reported.


Inhibition of water absorption in the intestine by Staphylococcus aureus delta-toxin. Water absorption in intestinal segments was monitored by measuring the concentration of polyethylene glycol, a nonabsorbable reference marker, in a balanced salt solution continuously perfused through the lumen. Staphylococcus aureus delta-toxin inhibited water absorption in rabbit jejunum and ileum perfused in vivo and in guinea pig ileum perfused in vitro. Cholera toxin also interfered with water absorption in guinea pig ileum maintained in vitro, but S. aureus enterotoxin B had no demonstrable effect on this tissue.


Demonstration of the cardiotoxicity of the thermostable direct hemolysin (lethal toxin) produced by Vibrio parahaemolyticus. Intravenous injection of the thermostable direct hemolysin (lethal toxin) produced by Vibrio parahaemolyticus caused rapid death of rats. Studies by electroencephalography and electrocardiography showed that after intravenous injection of the toxin the electroencephalogram remained normal for quite a long time after the heart of the animals had stopped beating. Depression of intraatrial and intraventricular conduction of electrical activation, including atrioventricular block, was observed in electrocardiograms of animals injected with the toxin. The toxin was also found to be toxic to cultured mouse heart cells. When it was added to the medium, the beating rhythm of the cultured heart cells increased temporarily and then soon stopped abruptly. The effect of the toxin on cultured mouse heart cells was blocked by preincubation of the toxin with a ganglioside mixture. From these results it is concluded that the thermostable direct hemolysin (lethal toxin) had cardiotoxic activity, and thus administration of the toxin causes rapid death of animals.


Effect of penicillin on the succession, attachment, and morphology of segmented, filamentous microbes in the murine small bowel. Indigenous segmented filamentous microbes attach to murine ileal epithelial cells. These microbes can be seen on the epithelial surface with a scanning electron microscope. They colonize preferentially the distal ileum in mice. Penicillin, placed in the animal's drinking water, eliminates the microbes from the mouse ileum, but recolinization of the ileum is observed 4 to 5 weeks after the penicillin treatment is stopped. Within 3 to 5 h after rats are given penicillin, the morphology of the microbes is changed. Their external surfaces are wrinkled or broken. Vacated and partially vacated attachment sites are observed. Almost all of the organisms disappear from murine ilea after the animals are exposed to penicillin for 10 h. These observations are discussed in relation to the microbe itself and in its interaction with ileal epithelial cells.


Distribution of cholera organisms in experimental Vibrio cholerae infections: proposed mechanisms of pathogenesis and antibacterial immunity. This study was undertaken to determine the sequence of events in the microenvironment of the intestinal tract that culminate in the symptoms of cholera and to attempt to define more clearly the mechanisms involved in antibacterial immunity. The extent to which mucus occurs in the normal intestine of rabbits and the appearance of the intestinal villi in unfixed frozen sections was demonstrated. The villi and intervillous spaces were found to be normally covered by a layer of mucoid material that formed a mucous zone between the intestinal contents and the tips of the villi. The distribution of cholera organisms in normal and immunized animals was demonstrated by the staining of frozen-tissue sections with specific fluorescent antibody. Study of tissue sections from normal animals showed that the onset of fluid accumulation was concomitant with the establishment of large masses of organisms in the intervillous spaces and crypts of the intestine after the successful penetration of this mucous zone. Tissue sections from animals actively or passively immunized against a cell wall antigen of Vibrio cholerae showed clumping of vibrios in the lumen and restricted distribution in the lumen and luminal border of the mucous zone. Antibody was not lytic in vivo.


New adjuvants on a polymethylmethacrylate base. A new type of adjuvant using influenza virus as an antigen is presented. The new adjuvant was produced by polymerizing monomeric methylmethacrylate in the presence of the antigen. As a comparison, influenza virus was added to previously polymerized polymethylmethacrylate particles. In animal experiments, the antibody response in mice and guinea pigs was measured. After polymerization in the presence of the antigen, the adjuvant effect was dependent on the methylmethacrylate concentration used, reaching an optimal concentration at 0.5%. This adjuvant preparation was considerably more effective than simple addition of virus to comparable polymethacrylate preparations or to aluminum hydroxide. The latter two adjuvants were approximately equivalent in effectiveness.


Mycoplasma pneumoniae in hamster tracheal organ culture studied by scanning electron microscopy. Hamster tracheal rings in organ culture were inoculated with a virulent strain of Mycoplasma pneumoniae and examined by scanning electron microscopy. A progressive increase in epithelial cell injury was detected from 48 to 96 h post-inoculation and was characterized by apparent loss of the apical portion of ciliated cells. M. pneumoniae attaching to the epithelial cell surfaces could be identified by comparison with the surface morphology of mycoplasmas grown on glass cover slips.


Ultrastructural observations on Ehrlichia equi organisms in equine granulocytes. The ultrastructure of the etiological agent of equine ehrlichiosis, Ehrlichia equi, was studied in equine peripheral leukocytes. The organisms occurred within membrane-lined cytoplasmic vacuoles of neutrophils and eosinophils. Ovoid, round, and rod-shaped profiles were observed. From 1 to 33 organisms were present in a thin-section profile of a cytoplasmic vacuole. Many cells contained multiple organism-containing vacuoles. The organisms had a cell wall and plasma membrane, and internally they consisted of electron-dense and lucid areas. A great variation in size was observed. The morphological features were most consistent with agents of the genus Ehrlichia.


Aleutian disease of mink: production of 14C-labeled antiviral antibodies by mink lymphoid cells in vitro. Lymphoid cells from mink infected with Aleutian disease virus produced labeled, specific antiviral antibody when incubated in medium containing [14C]tyrosine.


Ganglioside and rabbit erythrocyte membrane receptor for staphylococcal alpha-toxin. The hemolytic activity of staphylococcal alpha-toxin is inhibited by an N-acetylglucosamine-containing ganglioside (GlcNAc-ganglioside) but not by any of the related glycolipids. The GlcNAc-ganglioside also is precipitated with the toxin by a gel-diffusion technique. It is postulated that GlcNAc-ganglioside may be the membrane receptor for the alpha-toxin.


Cell-mediated immune response to influenza virus infections in mice. The local and systemic cell-mediated immune (CMI) responses to influenza virus infection in mice were examined by leukocyte migration inhibition and lymphocyte-mediated cytotoxicity tests. Mice were inoculated intranasally with 5 50% lethal doses of the A/WSN (H0N1) strain of influenza virus. Cells from the lymph nodes draining the upper and lower respiratory tract were used to measure the local response, and the spleen was the source of cells used for systemic determinations. The local response by pulmonary lymph node cells was greater and appeared earlier than was observed systemically in the spleen. The specificity of the CMI response was investigated by using a heterologous virus strain, A/Jap (H2N2), and recombinants A/Jap-NWS (H2N1) and A/NWS-Jap (H0N1), obtained from a cross between A/Jap (H2N2) and a virus, A/NWS (H0N1), with surface antigenic specificity similar to that of the inoculated virus. From the results of both tests used as correlates of CMI, it appeared that the response was specific against the hemagglutinin component of the inoculated virus. No reactivity was observed against the heterologous virus A/Jap (H2N2) nor against the recombinant A/Jap-NWS (H2N1) bearing the same neuradminidase as that of the inoculated virus.


Comparative production of interferon by explanted lymphoreticular tissue and alveolar macrophages from rabbits and humans. Studies were undertaken to compare interferon production among a variety of lymphoreticular cells, with emphasis on the alveolar macrophage. Explanted cells from rabbit lung, spleen, peritoneum, bone marrow, and blood produced interferon in varying amounts in response to six of the seven viruses studied. The various lymphoreticular tissues responded differently to a single interferon-inducing virus, and each tissue produced varying amounts of interferon when stimulated by different viruses. In addition, glass-adherent rabbit alveolar macrophages produced more interferon than did the nonadherent subpopulation. Human blood and lung cells produced much less interferon than did the equivalent rabbit cells under similar conditions of stimulation. It appeared that interferon production may have been controlled by several variables, including the species, the type of inducer, and the type of tissue and cell.


Quantitative reduction of 2,3,4-triphenyl tetrazolium chloride by hamster trachea organ cultures: effects of Mycoplasma pneumoniae cells and membranes. The ability of Mycoplasma pneumoniae cells and membranes to affect tetrazolium reduction by hamster trachea organ cultures was evaluated. Uninfected trachea explants reduced 2,3,5-triphenyl tetrazolium chloride (TTC) and nitro-blue tetrazolium when incubated at 37 C in the absence of air. Reduced tetrazolium salts (formazans) were extractable with acetone or ethylene glycol and could be quantitated spectrophotometrically. The optimal assay system involved the use of three or more tracheal rings incubated for 2 h in 0.12% TTC in Tyrode balanced salts supplemented with 1.2% sodium succinate. Formazan was extracted for 5 min with acetone, and the optical density (490 nm) was determined. Trachea explants with metabolic activity reduced or obliterated by freeze-thaw lysis, heat (56 C X 30 min), or cyanide (0.1 M NaCN X 30 min) had negligible ciliary activity and tetrazolium reduction activity (optical density at 490 nm [dry weight]). Tracheas exposed to mycoplasma cells or membranes also showed significantly decreased ciliary activity and tetrazolium reduction; e.g., only 5pc of the ciliary activity and reduction capacity remained after 5 days in culture when infected with M. pneumonia PI 1428 cells. The data indicate that the exposure of ciliated respiratory epithelium to mycoplasma cells or membranes results in diminished oxidative metabolism, and that the ability to reduce TTC to its formazan is correlated with relative ciliary activity.


Hydrolysis of urea in the gastrointestinal tract of "monoxenic" rats: effect of immunization with strains of ureolytic bacteria. Axenic rats, in whose feces urea is ordinarily excreted, were inoculated with ureolytic strains of Lactobacillus or Actinobacillus originally derived from the microflora of "holoxenic" rats. In these "monoxenic" animals, harboring one or another of the bacterial strains, fecal urea was hydrolyzed, with a more rapid onset of ureolysis in the case of Actinobacillus as compared with Lactobacillus. In vitro, a parallel difference between the two strains with regard to the onset of ureolysis was observed, hydrolysis beginning at the onset of growth in the case of Actinobacillus and only at the end of the exponential growth phase in the case of Lactobacillus. Extracellular urease activity was demonstrated in cultures of Lactobacillus, whereas none was found extracellularly with Actinobacillus. The pH optimum for the Lactobacillus urease in vitro was found to be 3.0, whereas the corresponding value for Actinobacillus was 6.0. In the two types of monoxenic rats, urea was consistently present in the small intestine and virtually absent from cecum and colon. Hydrolysis of urea in stomach was almost complete in rats bearing Lactobacillus but much less so in animals monoxenic with Actinobacillus, despite essentially equal numbers of organisms in that location. When rats carrying a monoflora of ureolytic Lactobacillus were immunized with either whole cells or soluble extract of the same organism, urea appeared in cecum and feces, indicating suppression of ureolytic activity. Immunization with an extract of nonureolytic Lactobacillus failed to produce such a result. Similar immunization techniques applied to animals monoassociated with ureolytic Actinobacillus did not alter ureolysis, and no appreciable quantity of urea appeared in feces. These studies demonstrate that it is indeed possible to inhibit the ureolytic activity of some bacteria in vivo by immunological means, but that the urease system of other organisms may not be as amenable to such manipulation.


Immunosuppressive activity of concanavalin A. Daily intraperitoneal doses of concanavalin A (Con A) produced a dose-related inhibition of adjuvant-induced arthritis in rats. Con A was also effective on established arthritis, markedly relieving the disease after only three doses. The inhibitory effect of Con A was neutralised by pre-incubation with ovalbumin, although this treatment did not modify the delayed phlogistic action of Con A in rat paws.


Adrenaline and tropical pulmonary eosinophilia. Injections of adrenaline produced an immediate rise in peripheral absolute eosinophil count in cases of tropical pulmonary eosinophilia, but a fall or no change in other conditions with similar clinical manifestations. A similar early eosinophilic effect was noted in control guinea pigs as well as in Ascaris larvae-fed guinea pigs. The effect of adrenaline on eosinophil counts in cases of tropical pulmonary eosinophilia was not paralled by effects on total erythrocyte or leucocyte counts or on packed cell volumes. The early eosinophilic effects of adrenaline in guinea pigs was blocked by pehntolamine but no be propranalol. Adrenaline-induced changes on eosinophil counts may be used as a disgnostic test of tropical pulmonary eosinophilia.


The alkaline phosphatases of human liver: an immunochemical study. Four out of 18 human liver alkaline phosphatase (AP) preparations reacted with anti-liver and anti-intestinal AP. The liver AP that reacted with anti-intestinal AP, designated as intestine-like AP, was precipitated at 50% ammonium sulfate whereas the major liver AP precipitated at 66% saturation. Gel filtration showed that liver AP, intestine-like AP and intestinal AP contained AP with apparent molecular weights of 130,000 daltons; the intestine-like AP contained a second but smaller component of 70,000-80,000 daltons. AP extracted from intestine also contained this smaller component; its electrophoretic mobility was that of an a2-globulin, whereas that of the intestine-like AP had a mobility of beta-globulin. The similarity of the intestine-like AP to the AP variant found in hepatomas is stressed.


Experimental hypersensitivity pneumonitis. Humoral and cell-mediated immune response of cattle to Micropolyspora faeni and clinical response to aerosol challenge. Hypersensitivity pneumonitis was produced in calves, following repeated exposure to aerosols of Micropolyspora faeni both with and without prior systemic sensitization. The condition was associated with rising serum, nasal and broncho-alveolar antibody titers and with transient ability of peripheral lymphocytes to synthesize macrophage migration inhibition factor. Clinical response to aerosols of M. faeni suggest participation of reaction types, I, III and IV of Gell and Coombs.


Discrepancy between skin reactivity and blastogenic activity of fractionated PHA-P. Firstly, it was reported that phytohemagglutinin P (PHA-P) can induce the immediate skin reaction besides the delayed one. 70 out of 80 cases (87.5%) gave positive immediate reactions with 1 mug of PHA-P. There was a close correlation between the radioallergosorbent test (RAST) counts against PHA-P and the total IgE value or erythematous diameter of the immediate skin reactions. Absorption with anti-IgE or PHA-P reduced the RAST counts against PHA-P to 79 and 56% of their initial values, respectively, but did not effect the RAST counts against steptokinase-streptodornase (SK-SD). These results suggest that the immediate skin reaction might be induced by specific IgE antibody against PHA-P. Secondly, using three fractions of PHA-P obtained by Sephadex G-200 gel filtration, skin test and RAST in humans, ACA in rats, and 3H-thymidine incorporation into human leukocytes, were carried out and data obtained that showed some discrepancy between them. That is, on skin testing, it was shown that all of the three fractions (fr.) had the ability to elicit the immediate and delayed skin reaction in humans and rats with the order of fr. 1, 2 and 3, and it was confirmed by RAST. On the other hand, the leukocyte-stimulating activity resided in fr. 1 and 2, while fr. 3, which seemed to be a group of subunits with a molecular weight of around 35,000, had almost no activity. These findings have been discussed in view of the earlier studies on the structure of PHA-P


A comparison of the anaphylactoid actions of a synthetic linear dextran and a natural branched dextran. Synthetic linear dextran of molecular weight 40,000 produces the systemic anaphylactoid reaction in rats although it is about 4 times less active than the natural branched dextran of similar molecular weight. Similarly, it is less active on local injection into the foot or skin. However, in rats which have been bred for non-reactivity to systemic dextran, it is more active on local injection, resembling the activity of a branched natural dextran of much lower molecular weight (6,000). In human skin, the synthetic linear 40,000 sample is also more active than the natural branched sample of similar molecular weight in producing a wheal and erythema. The results suggest that the dextran receptor in the skin of man may be similar to that in rats genetically resistant to systemic dextran.


Effect of D2O on phytohemagglutinin-induced lymphocyte proliferation. The effect of D2O on phytohemagglutinin (PHA)-induced proliferation of human lymphocytes in vitro was examined. A significant dose-dependent inhibition of the lymphocyte reactivity by D2O was observed. This inhibition did not require the simultaneous presence of D2O and PHA in the cultures. Both cytotoxic and cytostatic mechanisms for this inhibition are implicated.


Direct macrophage migration inhibition test in DNCB contact sensitized guinea pigs. Using dinitrochlorbenzene contact-sensitized guinea pigs, several DNP conjugates have been assayed in the direct macrophage migration inhibition test (MMIT). Although no significant differences could be observed between the carriers used, conjugates prepared from  serum proteins and epidermal extracts tended to give the strongest inhibition of macrophage migration. Conjugates prepared from cells cultured in vitro in the presence of hapten did not cause more inhibition than control conjugates prepared in the absence of cell metabolism. The direct MMIT showed statistical differences between sensitized and nonsensitized groups of guinea pigs. However, none of the conjugates permitted conclusions to be drawn with regard to individual animals.


Tumor-bound immunoglobulins. I. Further analysis of the characteristics of binding of immunoglobulins to in vivo-grown tumor cells. Immunoglobulin (Ig) "coating" on different in vivo growing mouse tumors was investigated using several approaches. Radioiodine-labelled purified protein A from Staphylococcus aureus, a specific reagent for Fc of IgG, and/or iodinated purified antibodies against mouse immunoglobulins, were fixed by various in vivo-grown mouse tumor cells, but not by the corresponding in vitro-cultivated tumor cells. Removal of host macrophages from the in vivo tumor-cell preparations did not affect the fixation of anti-mouse Ig reagents by the tumor cells. After an initial lag period in vivo, the intensity of the Ig coating of tumor cells increased with time after tumor inoculation. Conversely, the detectable coating decreased rapidly as a function of time after ascites tumor cells were explanted in vitro at 37 degrees C. Iodoacetamide did partially block this 37 degree C in vitro-induced "uncoating process". Surface-bound Ig could also be released from in vivo-coated tumor cells treated at low pH in vitro. Analysis of the behavior of tumor-bound Ig indicated a composite pattern. At the membrane level, uncoating was best shown with in vitro incubation favorable to cellular metabolism; however IgG was detected equally well in the supernatants of the cells, whether they were incubated at 37 degrees C or at 4 degrees C, and so far has failed to display antibody activity towards uncoated tumor cells. This was in contrast to the case of IgG released from ascites tumor cells cultured at low pH in vitro: such eluted Ig, when neutralized, could rebind to the "same" uncoated tumor cells. Under favorable metabolic conditions, the fate of tumor-bound Ig might be one of internalization and/or of degradation.


Biochemical changes during regression and regrowth of hormone-dependent GR mouse mammary tumors. Discontinuation of recommencement of estrone-progesterone treatment causes regression and regrowth, respectively, of transplantable hormone-dependent GR mouse mammary tumors. This tumor model was found convenient for the demonstration of hormonal responses in hormone-dependent mouse mammary tumor cells. Tumor regression was palpable 2 days after discontinuation of hormonal treatment and tumors reached half their size within 3-6 days. At this point, hormones were readministered and 2-4 days later the tumors had grown to their preregression size. In the growing and regressing tumor we have investigated: (1) the content of RNA, DNA and protein per g wet weight, (2) in vivo incorporation for 45 min of 32P-orthophosphate into RNA and DNA and the 35S-methionine incorporation into protine; (3) SDS polyacrylamide gel electrophoresis of soluble proteins after in vivo incorporation of 14C- and 3H-leucine. The RNA content per g wet weight was found to decrease during regression and to increase during regrowth. DNA and protein content showed no variation. RNA/DNA ratio thus varied in parallel to the RNA content. The precuursor incorporation into all three macromolecular species decreased during regression. The incorporation into DNA showed the most pronounced decrease. After readministration of hormones, regrowth was accompanied by a rapid increase in precursor incorporation into RNA, while the incorporation into DNA showed a lag of 1 to 2 days. SDS polyacrylamide gel electrophoresis was carried out with soluble proteins labelled in vivo with 14C- and 3H-leucine during regression and regrowth, respectively. No differences in 14C/3H ratios could be demonstrated for the major fractions detectable in the electropherogram.


Detection of private and common tumor-associated antigens in murine sarcomas induced by different chemical carcinogens. Two fibrosarcomas of similar histological type, induced in C3Hf mice by either methylcholanthrene or 3,4-benz(a)pyrene, were shown to have individually unique tumor-rejection antigens in classical transplantation-type experiments. By contrast, sera of autochthonous mice, which resisted only transplants of the immunizing sarcoma, were found to contain complement-dependent cytotoxic antibodies, specific for both sarcomas, in vitro. The existence of individually unique as well as common tumor-associated antigens in chemically-induced murine sarcomas is suggested. The private "tumor transplantation-type" antigens elicited tumor rejection responses in vivo. The common tumor-associated antigens, although immunogenic in autochthonous hosts, inducing the production of tumor-specific antibodies, failed to induce transplantation cross-resistance in vivo. This study supports the contention that, in carcinogen-induced murine tumors, and perhaps in human neoplasms as well the evaluation of humoral (and cell-mediated) immune responses in vitro may not reflect tumor rejection-type immune responses in vivo.


In vitro detection of cellular immunity to melanoma antigens in man by the monocyte spreading inhibition test. In vitro inhibition of monocyte spreading (a correlate of cellular immunity) was used to detect cell-mediated immune reactions of melanoma patients to specific melanoma antigens. Two soluble preparations of human melanoma antigens (MA-1 and MA-2) and one of a breast carcinoma (BCA) were prepared. The preparations were incubated in vitro with mononuclear cells isolated from the blood of 24 patients with melanoma, six patients with malignancies other than melanoma and 14 healthy donors. Spreading of monocytes from healthy donors was not inhibited by either MA-1 and MA-2 or BCA. MA-1 and MA-2 significantly inhibited the spreading of monocytes from patients with melanoma, while monocytes from patients with other malignancies were not affected. Spreading of monocytes from patients with melanoma was inhibited by the preparation of BCA. We conclude that inhibition of monocyte spreading can detect, in vitro, a cellular immune reaction to specific melanoma antigens in patients with melanoma.


In vitro cell-mediated immune reactions of melanoma and colorectal carcinoma patients demonstrated by long-term 51chromium assays. In a series of consecutive 51chromium (51Cr) assays, 21 melanoma and 14 colo-rectal carcinoma patients were tested for their in vitro cell-mediated immune reactions to melanoma, rectal adenocarcinoma, and normal fibroblast target cells. Blood lymphocytes (BL) from four individuals were included in each experiment. In 9/14 experiments the BL effects of 2 melanoma patients were compared to BL effects of 2 colorectal carcinoma patients. In two experiments, BL from 1 melanoma patient and 1 colorectal carcinoma patient were compared for cytotoxic effect with each other and with BL from 2 normal healthy donors, and in the remaining 3 experiments BL from 2 melanoma patients were compared with BL from 1 healthy donor and one patient bearing a tumor which was neither a melanoma nor a colorectal carcinoma. Eleven out of 14 experiments were performed in a criss-cross manner. Target cells in the first three tests of this series consisted of tumor cells and fibroblasts explanted from a single melanoma patient. In all of the remaining experiments, each BL population was tested for cytotoxicity against both a tumor-fibroblast target cell pair explanted from one of two melanoma patients and a tumor-fibroblast target cell pair explanted from a rectal adenocarcinoma patient. Out of 35 tumor patients, 27 (77%) demonstrated a selective cytotoxic effect on the tumor target cells compared to the corresponding fibroblasts, while 4 patients (11%) showed a selective effect on the fibroblasts, and 6/29 patients showed a selective effect on the other type of tumor cells compared to matching fibroblasts. In 8/11 experiments (including two repeat tests) tumor-specific BL effects were demonstrated in a criss-cross manner. BL separations and 51Cr tests were repeated in 11 of the 35 patients 2-4 weeks after their original tests. BL populations from these 11 patients reproduced their individual earlier effects, whether these effects showed specific, non-specific, or no cytotoxicity. In each assay, differences in sensitivity between fibroblasts and tumor target cells in matched pairs were analyzed by comparing the effects of BL from the two controls. No differences in target-cell sensitivity could be demonstrated.


Viral "tumorigenesis" in man: cell markers in condylomata acuminata. Determination of the glucose-6-phosphate dehydrogenase (G-6-PD) phenotype of a neoplasm occurring in a heterozygous female can be used to trace the cellular origin of the tumor. This technique was performed on 834 individual verrucous subunits from four condylomata acuminata (venereal warts) arising in two patients heterozygous for a B and an A gene at the G-6-PD locus. All four specimens contained both A and B types of G-6-PD. Furthermore, even single verrucous subunits from each specimen occasionally contained both enzyme types. These data indicate that condylomata acuminata have a multicellular origin. The initial number of cells which, after viral infection, developed into a condyloma acuminatum was estimated to be about 4,400 cells, on the basis of statistical analysis of the data in one case.