Binding of latent rheumatoid synovial collagenase to collagen fibrils. Collagenase released from rheumatoid synovial cells in culture is in a latent form. Subsequently, it may be activated by limited proteolysis. This study was designed to determine whether latent enzyme could bind to collagen fibrils and await activation. The data showed that latent collagenase bound to fibrils equally well at 24 degrees C and 37 degrees C, but that this represented little more than half the binding achieved by active enzyme at temperatures lower than that at which fibrils can be degraded. Binding was not inhibited by the presence of alpha2 macroglobulin, the principal proteinase inhibitor of plasma which cannot complex with inactive or latent collagenase but readily complexes with active species of enzyme. The data support the hypotheses that inactive forms of collagenase accumulate in tissues by binding to substrate, and that activation by proteases such as plasmin initiates collagen breakdown.


Mass spectrometric analyses and characterization of Kepone in environmental and human samples. A specific portion of our environment has been contaminated with Kepone, or chlordecone. Additionally, some specific human exposures to high concentrations of Kepone have been confirmed. Gas chromatography mass spectrometry involving chemical ionization and high resolution mass spectrometry were used to detect, identify and confirm the presence of Kepone, Kepone photoproducts and a reduction product of Kepone in environmental and human samples. Field desorption, field ionization and electron impact mass spectrometric methods, as well as infrared and nuclear magnetic resonance techniques were used to characterize and identify Kepone hydrate and hemiketal in benzene and methanol solutions, respectively.


[Modified method of differential staining of sister chromatids]. A modified method of obtaining differential staining of sister chromatids is described. It is simple, rapid, and effective, and at the same time inexpensive and accessible, since it allows one to use available reagents. When 5-bromdeoxyuridine was administered 24 hours before fixation into the Chinese hamster cell culture the percentage of metaphases with a differential chromatid staining constituted 95--98, and when this substance was administered 28 hours before fixation into the human lymphocyte culture this percentage varied from 75 to 92, depending on the individual. The mean number of sister chromatid exchanges in human lymphocytes failed to depend on the time of fixation.


Urodynamics in benign prostatic hypertrophy. Synchronous urinary flow and pressure studies were carried out on 51 male subjects of whom 12 were normal subjects and 39 had benign prostatic hypertrophy with varying degrees of bladder outlet obstruction. A urodynamic scoring system was evolved for accurate and objective diagnosis of bladder outlet obstruction. The minimum urethral resistance was found to be the most valuable single urodynamic parameter for the diagnosis of bladder outlet obstruction. Hitherto this urodynamic parameter was determined through tedious calculations. In the course of the present study a new instrument, the Urethroresistance, was devised for the direct recording of urethral resistance during micturition.


Biochemical studies of trophic dependences in crayfish giant axons. Data from previous histological studies indicate that long-term survival of crayfish medial giant axons might be due in part to trophic support from cells of the surrounding glial sheath which often hypertrophy in response to transection of the medial giants. The biochemical studies reported herein show that segments from transected ventral nerve cords (VNC) always incorporate more [3H]leucine into protein than do corresponding segments from intact VNCs. Furthermore, the relative amount of [3H]leucine incorporation in severed segments seems to be influenced by distance and direction from the lesion site as well as time after lesioning. Similar spatiotemporal parameters were previously shown to be correlated with extent of glial hypertrophy around severed medial giant axons. Quantitative autoradiography of medial giant axons after incubation in [3H]leucine revealed that the grain density of label in glial sheaths surrounding severed medial giants was over two-fold greater than in sheaths around corresponding control axons. Moreover, the grain density in the axoplasm of severed medial giants was nearly four-fold greater than the grain density in the axoplasm of control axons. Data from experiments using short or long labeling intervals suggests that labeling in the medial giant axoplasm may be due more to transfer from glial sheath cells than from inherent axonal synthetic mechanisms. In light of this and other data, we concluded that long-term survival of severed medial giant axons is probably due to the direct transfer of trophic substances from cells of the glial sheath into the axon.


Characterization of an antiserum to synaptic glomeruli from rat cerebellum. Rabbit anti-rat cerebellar synaptic glomeruli antiserum when absorbed with non-neural tissues reacts only with neural tissues when tested by indirect immunofluorescence on tissue sections. Further absorption with forebrain results in the antiserum which detectably reacts only with synaptic glomeruli and soma of Purkinje cells of both rat and mouse. The developmental expression of the synaptic glomeruli antigen(s) parallels the formation of synapses between mossy fibers and granule cells. Immature synaptic contacts do not contain recognizable antigen(s), whereas only at postnatal Day 15 glomeruli become antigen-positive. At this stage antigen in Purkinje cells is no longer carried in their dendrites, but becomes confined to the cell soma. Staggerer mutant mice still express the immature pattern of antigen distribution on postnatal Day 18.


Combination therapy of advanced head and neck cancer: induction of remissions with diamminedichloroplatinum (II), bleomycin and radiation therapy. Patients with unresectable, previously untreated head and neck cancer were given cis-diamminedichloroplatinum (II) (DDP), 3 mg/kg, with mannitol diuresis (day 1), followed by a continuous infusion of bleomycin, 0.25 mg/kg/day, days 3 through 10, after an initial loading dose of 0.25 mg/kg by rapid IV injection on day 3. The DDP was repeated on day 22, following which radiotherapy was delivered using standard doses, fractionations and portals. Patients were evaluated for response on day 22 and again at the conclusion of radiotherapy. Of 21 patients evaluable at day 22, there were four CR and 11 PR (greater than 50% reduction of all measureable disease), for a major response rate of 71%. Of five MR, four showed 30-60% reduction at the primary site. Of 16 who have finished the radiation phase of treatment, there are six CR, five PR and one MR with durations four to eight months. Toxicity in 33 patients included vomiting (33), alopecia (33), WBC less than 3000 (five), platelets less than 100,000 (one), dose-limiting mucositis during bleomycin (six) and peak serum creatinine greater than 2 (five), with one fatality. The regimen thus appears promising as initial therapy for the previously untreated patient. The same chemotherapy has produced much less encouraging results in prviously treated patients.


Microscopie-controlled excision of cutaneous tumors: chemosurgery, fresh tissue technique. A modification of Mohs' chemosurgery which eliminates the zinc chloride paste but follows the careful piece by piece excision, marking, and mapping has been developed for the treatment of cutaneous tumors. The technique is called Microscopic Controlled Exicision (MCE). This 8-year retrospective study reports a 97.2% cure rate for 532 determinant lesions. On hundred eighty-five (35%) of these cases followed for 5 years or longer had a 96% cure rate. The modified technique eliminates the pain of zinc chloride fixative paste, shortens the time required to perform the surgery, removes less uninvolved, normal tissues, and the final defect is a fresh surgical wound which can be repaired immediately. These advantages become highly significant since a high 5-year cure rate can be obtained. It is recommended that this technique be used for all tumors which recur after routine treatment by excision, radiation therapy, curettage and desiccation, or cryosurgery, for tumors with poorly defined clinical borders, for sclerosing basal cell epitheliomas, and for primary cutaneous carcinomas in areas which have a predilection for recurrence.


Detection of viral-like cores from the urine of patients with genito-urinary malignancies. Simultaneous detection assays on the core structures derived from the cerebrospinal fluid samples of patients with various types of central nervous system tumors have demonstrated the feasibility of this technique in detecting some of the diagnostic features of RNA tumor viruses. Similar assays done on urine samples from patients iwth various types of tumors in their genitourinary tracts have shown that of the 18 such samples from tumor patients, 15 or 83% were found to be positive. The control samples consisted of three from patients with benign prostatic hypertrophy and four from normal persons. None of these gave a positive reaction. [3H]DNA probes synthesized from the core structures from them hybridized readily to their corresponding polysomal RNAs but no to control tissues. The densities of particles from these samples have been found to be 1.168 g/ml for bladder carcinoma and 1.165 for prostatic carcinoma, the same densities as those found RNA tumor viruses.


A new methods for the 3'-deoxygenation of butirosins A and B. An aziridine ring-formation involving the reaction of adjacent amino and alcohol groups with triphenylphosphine, carbon tetrachloride, and triethylamine was applied at the 2' and 3' positions of butirosin A (1a) and B (1b). The amino groups at the 2' position of 1a and 1b were p-methoxybenzylated to increase the nucleophilicity of the nitrogen atom and to avoid the formation of a P-N linkage, and the N-p-methoxybenzyl derivatives were converted into the aziridine derivatives, which were then subjected to hydrogenolysis and removal of the protecting groups to give 3'-deoxybutirosin A (7a) and B (7b), respectively. This new method is compared with the conventional N,O-protecting method that involves several complex steps.


Origin of the membrane for the acrosomal process: is actin complexed with membrane precursors? The formation of the acrosomal process requires an increase of 40-150% in the plasma membrane limiting the cell body. In freeze fracture replicas and thin sections this new membrane behaves as a typical unit membrane. Since in sperm there are no vesicles or surface projections which could account for the increase in surface, we suggest that the new membrane is generated from precursors stored with the actin in the periacrosomal region. Consistent with this suggestion is the observation that compounds such as didansyl cysteine and N phenyl-1-napthylamine which fluoresce when they enter a hydrophobic environment stain the periacrosomal material.


Proliferative capacity constant of metazoan cells in culture. Proliferative capacity of metazoan cells in culture may be defined quantitatively by using GPAG-supplemented medium. For this purpose an expression called proliferative capacity constant (KPC) was introduced. KPC represents the logarithm of GPAG concentration over which the mitotic activity of cells is induced.


Differentiation of malignant neuroblastoma cells: evidence for a mitochondrial role. Morphological differentiation has been induced in vitro in mouse neuroblastoma cells by quinidine, dinitrophenol and dicoumarol. These agents are known to release calcium from mitochondria. Drugs acting in this way might be exploited in cancer therapy.


Repolarization abnormalities in survivors of out-of-hospital ventricular fibrillation. Survivors of out-of-hospital ventricular fibrillation (VF) are at high risk for recurrent VF, probably reflecting continued myocardial electrical instability. In this study 12-lead ECGs of 125 VF survivors with coronary heart disease were examined and compared to those of 98 ambulatory post-MI patients. The study was part of an effort to define clinical identifiers of patients likely to develop sudden cardiac death. Ventricular fibrillation survivors were commonly had premature ventricular complexes (PVCs):30% versus 13% (P less than 0.01). In addition, ECGs of VF survivors showed a significantly greater prevalence of ST-segment depression (46% versus 10%), T wave flattening (52% versus 26%), and QTc prolongation (35% versus 18%). It is proposed that these repolarization abnormalities represent asynchronous repolarization, which together with frequent PVCs, may set the stage for re-entrant ventricular dysrhythmias and ultimately VF. It is also possible that repolarization abnormalities together with premature ventricular contractions might serve as markers of patients with coronary heart disease who are at increased risk for sudden cardiac death.


Relationships among ventricular arrhythmias, coronary artery disease, and angiographic and electrocardiographic indicators of myocardial fibrosis. This study was performed to determine the relationships among angiographic, hemodynamic, clinical, and electrocardiographic data and premature ventricular contractions (PVCs). Arrhythmias were analyzed by 24 hour Holter monitor in 244 patients evaluated for chest pain by coronary angiography and left ventriculography. Using a categorical linear model, the presence of myocardial fibrosis as indicated by both abnormal left ventricular contraction (LVC) and abnormal initial QRS on electrocardiogram was found to be the only independent predictor of both frequent and complex ventricular arrhythmias (P less than .0001). All other descriptors, including the number of diseased vessels (greater than or equal to 75% obstruction), were dependent upon abnormal LVC in their association with PVCs. When the right anterior oblique view of the left ventriculogram was divided into nine segments to allow automated quantiative analysis of LVC, the prevalence of frequent PVCs was directly related to the number of abnormally contracting segments. Of patients with 0 abnormal segments, 11% had greater than or equal to 2 PVC/hr, in contrast to 44%, 73% and 100% of patients with 1-3, 4-6, and 7-9 abnormal wall segments, respectively (P less than 0.01). A similar quantitative relationship was found between premature ventricular contractions and abnormal initial forces indicating previous myocardial infarction on the electrocardiogram.


Management of diskogenic pain using epidural and intrathecal steroids. The use of methylprednisolone acetate (Depo-Medrol) injected by the epidural or intrathecal route for the relief of diskogenic back pain with or without radiculopathy is an adjunct to conservative management useful when conservative measures fail and surgical treatment is under consideration. This is especially true when symptoms have been present for only a few months. Corticosteroids injected in the same manner seem to have little effect on patients with symptoms persisting for periods longer than 3 months or in patients treated previously by surgical methods.


Special topics in clinical pathology.

Special topics in clinical pathology. The following topics are discussed in this presentation: A. Recent advances in the use and interpretation and methodology of antibiotic susceptibility testing. B. Improvements in sample submittal to obtain accurate results from your laboratory. C. Staining blood, bone marrow, and cytology specimens in the office laboratory. D. Pathogenesis and differential diagnosis of lipemia. E. Differential diagnosis of abdominal effusions.


[Mechanism of action of synthetic heparinoids: results in patients with hypertriglyceridaemia (author's transl)]. The effect of two synthetic heparinoids, SP-54 and Depot-Thrombocid, on serum lipids was studied in 17 patients with primary hyperlipidaemia (types IIb, IV and V of Fredrickson) and 15 healthy persons. SP-54 had no lipid-lowering effect, neither on oral nor rectal suppository application. Intramuscular injection of Depot-Thrombocid, however, resulted in a decrease of serum triglyceride concentrations of maximally 50% after six hours. There was a marked increase in the activity of the two lipolytic enzymes, lipoprotein lipase and hepatic triglyceride lipase in plasma, as well as an increase in free fatty acids and an extension of thrombin time and PTT. Twenty-four hours after injection all values returned to pretreatment levels. Intramuscular administration of Depot-Thrombocid two or three times a week for seven weeks had no lasting effect on serum lipids. However, there were considerable side effects such as haemorrhagic diatheses, hair loss and thrombocytopenia.


Sclerotherapy of bleeding oesophageal varices by means of endoscopy. From 1.1.1969 up to 1.11.1977, 640 patients with hemorrhage from gastro-oesopheal varices were managed by sclerotherapy of the oesophageal wall. In 90% this method succeeded in stopping hemorrhage or preventing a new bleeding during the next four months. Only 43 patients of the total number were treated because of impending hemorrhage under precise indications. After two or three sessions of sclerotherapy the interval of control can be extended up to one years without new danger of hemorrhage. Overall mortality was 14.5%; main causes of death were liver coma, uncontrollable hemorrhage, mediastinitis and pyothorax.--If liver function improves, a porto-systemic-shunt is performed whenever possible.--416 = 65% of the patients are still alive; 50% longer than one year up to eight years. Thus sclerotherapy seems to be the method of choice in uncurable massive hemorrhage from varicosities from the oesophagus. It is indicated in patients with decompensated liver function, and whenever a shunt procedure is anatomically or clinically impossible or not advisable, too.


Changes in plasminogen levels, plasmin activity and activity of antiplasmins during endotoxin shock in dogs. Antiplasmin activity was studied during various stages of lethal canine endotoxin shock by means of assays utilizing a new chromogenic tripeptide derivative (S-2251 AB Kabi Peptide Research Division, Mölndal, Sweden). By applying a preincubation time of 30 sec and 5 min, respectively, 'immediate' and 'time-dependent' antiplasmin activities were determined. During shock gradually decreasing values of 'immediate' antiplasmin activity was observed. 'Time-dependent' antiplasmin activity also revealed a decreasing pattern, these changes, however, were less pronounced when compared with 'immediate' antiplasmin activity. The changes of plasma antiplasmin activities observed were accompanied by decreasing values of plasminogen and evidence of plasmin activity. alpha-Antitrypsin and alpha2-Macroglobulin quantitated with electroimmunoassay technique also revealed decreasing patterns during shock.


Observations on 1,000 Millin prostatectomies. 1,000 Millin prostatectomies were performed under general anaesthesia and normal conditions of blood pressure (without hypotension) over a period of 6 years. Observations were made as to the blood losses (Davillas and Miliaressis, 1972), the length of hospitalization, the various complications and the death causes in 17 patients (1.7%).


Two str1cturally similar haptens each induce a different inherited idiotype. When mice of the A/J strain were immunized with azobenzenearsonate (ABA) directly coupled to a protein, they produced antibodies that share an inherited cross-reactive idiotype (CRI). In the antigenic determinant that induces CRI, ABA is very probably coupled to tyrosine, and the structure that protrudes from the polypeptide backbone has two benzene rings and a molecular weight of 419. When this same structure is separated from the polypeptide backbone by a spacer of 99 daltons, it induces a different inherited idiotype (ABA-HOP-e) in the same strain of mice. Our data suggest that antibodies with the idiotype CRI recognize the terminal benzene ring and the azo group, but do not fit closely around the second ring structure. Antibodies with the idiotype ABA-HOP-e have fine specificity for both benzene rings. Both idiotypes are inherited and both are linked to genes controlling constant regions of the heavy chains.


Split unresponsiveness to the trinitrophenyl determinant. I. Manoeuvers which suppress either humoral or cell-mediated immune responses. 2,4,6-Trinitrobenzene sulfonic acid (TNBS) injected intravenously (i.v.) makes mice fully tolerant to the trinitrophenyl (TNP) determinant. Administration of in vitro TNP-labeled syngeneic erythrocytes or thymocytes renders mice unable to develop contact sensitivity to picryl chloride, while the humoral anti-TNP responses seem to be unaffected. The reverse was found after pretreatment of mice with TNP-labeled isologous IgG (MGG) since only anti-TNP antibody responses, but not contact sensitivity to picryl chloride, were significantly reduced. TNP-coupled macrophages given to animals suppressed both the cell-mediated and humoral responses, and this might be due to the presence on their surface of TNP-labeled cytophilic antibody. TNBS administered i.v. binds to circulating proteins and formed blood elements. Thus the split unresponsiveness affecting either humoral or cell-mediated compartments after the injection of TNP-MGG or of haptenated cells respectively, is presumably due to dissecting events which in vivo after the injection of TNBS, occur simultaneously. These results may be interpreted to indicate that split unresponsive states to TNP determinants are mediated two independent mechanisms which require different tolerogen presentations to be triggered.


Idiotype-specific neonatal suppression of phosphorylcholine-responsive B cells. The effect on neonatal anti-idiotypic suppression on the expression of B cells of the T15 clonotype has been investigated at the level of individual clonal precursor cells. The results indicate that B cells of the T15 clonotype are almost completely eliminated from the repertoire for four months after neonatal injection of allogeneic anti-idiotypic serum. The degree of this suppression is dependent on the amount of anti-idiotypic antibody administered and is less profound if anti-idiotypic antibody is given after the first week of life. No suppression was observed when anti-idiotypic antisera were administered to mice 30 days of age or older, which may indicate that immature B cells are the population most susceptible to suppression. However, since suppression could be reversed by administration of T15 myeloma protein several days after injection of anti-idiotype, the inability to suppress adult BALB/c mice may have been due to the high level of T15 idiotype normally present in their serum. Finally, phosphorylcholine-responsive B cells of identifiable clonotypes other than T15, even a clonotype sharing antigen-combining site determinants with T15, appear unaffected by anti-T15 suppression.


Evolution of the lymphoid system. II. Evidence for immunoglobulin determinants on all rainbow trout lymphocytes and demonstration of mixed leukocyte reaction. Indirect fluorescent antibody analyses utilizing antiserum specific for rainbow trout serum immunoglobulin (Ig) demonstrated that essentially all rainbow trout lymphocytes from thymus, spleen, anterior kidney and peripheral blood possess either Ig determinants or structures which cross-react with those determinants found on serum Ig. Peripheral blood leukocytes from rainbow trout were found to be caple of participating in a mixed leukocyte reaction. This observation is discussed in terms of the evolution of T cell function.


Reconstitution of natural cell-mediated cytotoxicity with specific antibodies. The apparent nonselective reactions of natural cell-mediated cytotoxicity (NCMC) are selective when tested by inhibition of cytotoxicity with competitor cells indicating a recognition of specificities by the effector cell. N cells that mediate this NCMC in humans have most of the characteristics of K cells that mediate antibody-dependent, cell-mediated cytotoxicity (ADCC) and possess Fc receptors. IgG antibodies attached loosely to N cells through their Fc region, form part of the class of lymphocytes with surface immunoglobulin. We hypothesized that ADCC and NCMS involved similar mechanisms but with the specificity of NCMC directed by the natural IgG antibodies already attached to N cells. Removal of the antibodies with trypsin and reconstitution with specific anti-HLA antibodies produced specific effector cells supporting the role of antibodies on N cells as directors of specificity in NCMC.


A serum-free medium for the Mishell-Dutton system. A serum-free medium which provides high recoveries of plaque-forming cells in the primary immune response to sheep erythrocytes is described. In this medium the serum was replaced by heated fetuin and by excess of nonessential amino acids. Glycopeptides, obtained by excessive pronase treatment of fetuin were partially capable of replacing the nonhydrolyzed fetuin.


On the synthesis of serine and homoserine samples asymmetrically labelled with tritium and deuterium in the hydroxymethylene group. (1R) [1-3H, 2H1] 3-Phenylpropanol, the key intermediate in the synthesis of (4R) [4-3H, 2H1] D,L-homoserine and of the (4S)-isomer, is obtained from (1S) [1-2H1] 3-phenylpropanol and (1RS) [1-3H] ethanol upon incubation with yeast alcohol dehydrogenase and NAD+; under similar conditions 2-phenylethanol undergoes very small exchange with [1-2H2] ethanol.


Effect of magnitude of daily continuous radiation dose on serum proteins and survival in rats. Changes in serum prealbumin, A1-macroglobulin, transferrin, and complement levels were studied in rats irradiated with daily doses of 100R (0.0258 C/kg), 200R (0.0516 C/kg) and 400R (0.1032 C/kg) until death. The daily dose of 100R applied for 39 days produced no changes in prealbumin and transferrin and a rise in the levels of A1-macroglobulin and complement. Shortly before death, i.e., between 39 and 44 days of irradiation, the levels of all four patients dropped. Daily doses of 200 and 400R caused a reduction in the levels of all four proteins since the very beginning. The LD 50 was 4 270R (1.1017 C/kg) at 100R/day, 4 060 R (1.0475 C/kg) at 200R/day, and 4 520 R (1.1342 C/kg) at 400R/day.


Heterogeneity of circulating human C-peptide. Values reported for serum C-peptide immunoreactivity in healthy individuals vary considerably. To assess factors that contribute to this finding, three human C-peptide assay systems were developed utilizing three distinct antisera that react differently with various C-peptide fragments. Preparations of natural pancreatic and synthetic human C-peptide standards were compared immunologically in these systems. The curves of the natural C-peptide and the synthetic preparations were not identical. The relative immunoreactivity of each standard varied, depending on the particular antiserum used. Serum C-peptide concentrations varied when measured in the different assay systems. Furthermore, the results of dilution and recovery tests and stability of the C-peptide during storage showed differences among the three assays. Gel filtration of serum indicated heterogeneity within the major C-peptide peak, and in addition, a smaller peak of lower molecular weight material was present in some samples. Although degradation of serum C-peptide may occur during storage, fragments of C-peptide may also be secreted or arise during in-vivo metabolism. Thus, the present studies indicate the need for careful standardization and checking of each particular assay system before its use in clinical studies.


Effect of mast cell degranulation on gastric mucosal damage produced by sodium taurocholate in the rat. The source of the histamine released during damage to the gastric mucosa has been investigated in rats using perfused total gastric pouches. Two groups of rats were treated with either intraperitoneal normal saline or compound 48/80, and agent that produces mast cell degranulation, over a 5-day period. On the 5th day, total gastric pouches were prepared and connected to a perfusion circuit that enabled a 20-ml volume to be circulated through the pouches. The experiments consisted of three 30-min periods during which transmucosal potential difference was monitored and ionic (hydrogen and sodium) flux measured; standard acid solution was used in the first two periods and a taurocholate solution in the third. Sodium taurocholate produced a significant increase in ionic flux and fall in the potential difference, the magnitude of the changes being similar in the 48/80- and saline-treated groups. Histamine was released from the mucosa in significantly greater amounts during the taurocholate period, and the increase was similar in both groups of rats. Histological examination of the stomachs confirmed mast cell degranulation in the 48/80-treated groups. We conclude that the histamine released during mucosal damage is probably derived from the "nonmast cell pool" and that this histamine may play a role in mediating the mucosal damage.


Detection of hepatocellular carcinoma during a clinical follow-up of chronic liver disease: observations in 31 patients. In 31 patients with an initial diagnosis of cirrhosis or chronic hepatitis hepatocellular carcinoma (HCC) was detected after a clinical follow-up of 8 months to 14 years with an average of 59 months. They had had no scintigraphic and biochemical abnormalities suggestive of HCC at the beginning. The follow-up period before the detection of carcinoma was shorter in patients positive for hepatitis B surface antigen compared with those negative for hepatitis B surface antigen. Analyses of clinical data during the follow-up and liver scans made shortly before tumor detection suggested that in most of these patients HCC became discernible relatively early in the course of cirrhosis or long before cirrhosis reached an advanced stage. A sharp rise in serum alpha-fetoprotein level proved highly diagnostic in 11, it remained low throughout in 7, and tumor was already unresectable in the majority. Although continuous and regular check for alpha-fetoprotein is imperative in patients with chronic liver disease, particularly in those with hepatitis B surface antigenemia, additional diagnostic tools are necessary for the detection of small HCC in its resectable stage.


[Use of nifedipine and nitroglycerin during exercise therapy in patients with myocardial infarct. Clinical and hemodynamic studies]. The hemodynamic effects of nitroglycerin and nifedipin and the application of both substances during a long term training program in patients with angina pectoris, myocardial infarction, and myocardial dysfunction (=48) were studied. Cardiac output, heart rate, and arterial blood pressure showed no significant changes after application of nitroglycerin and nifedipin. After nitroglycerin, however, there were significant drops of pulmonary arterial pressure and right atrial pressure during rest and exercise, whereas no changes were seen after nifedipin. With the application of nitroglycerin (but not with nifedipin) prior to the daily physical training program on a bicycle ergometer, all patients with angina pectoris and myocardial dysfunction showed an increasing training effect during the long term program and their angina pectoris improved.


[Antigen-elimination technic. An in vivo method for studies of the human immune response against horse gamma globulin]. In 20 patients treated with antilymphocyte horse gamma-globulin (ALG) the immune response was tested by means of the antigen elimination technique before, during and after ALG-therapy. The comparison of this method with agargel diffusion test, indirect hemagglutination and skin tests showed that immunization of patients against horse protein is indicated earlier and more reliable with the antigen elimination. Therefore this technique should be utilized during a therapy with foreign proteins especially when the results of other immunological test methods are insufficient.


[Cytoid bodies in the human skin].

[Cytoid bodies in the human skin]. Cytoid bodies represent roundish or polygonal, approximately cell-sized structures in histological sections. Such bodies have been reported to be of pathologic significance in several cutaneous disorders. Histological, immunological and electron microscopic investigations were performed in order to (1) characterize the different types of cytoid bodies in human skin, (2) elucidate their origin and nature and (3) determine their significance in cutaneous histopathology. A distinct identification of elastic globes, Russell bodies, Civatte bodies, amyloid bodies and cytoid fibrin bodies could be made. In addition, other substances such as nuclear or cytoplasmic debris, cytolytic fragments or basement membrane proliferations were found in form of cytoid aggregates in several dermatoses. However, none of the different groups of cytoid bodies was evaluated to be of pathognomonic significance for a specific disease. All of them are rather symptomatic for a number of anatomical or pathological principles and events in the dermo-epidermal junction area. Thus, the presence of cytoid bodies is not a major diagnostic aid in histopathology but may give valuable hints for a better interpretation of the morphogenesis of etiologically unrelated skin disorders.


Centromeric banding (C) of sequentially Q- and R-banded human chromosomes. A modified C-banding technique is described that produces C bands on human chromosomes after sequential Q and R banding and retains good chromosome morphology. Despite the considerable exposure to UV light during sequential Q and R bandings, clear C bands could still be achieved. Employing the present technique, Q, R, and C polymorphisms can be recorded on a single metaphase.


Structure and variability of human chromosomes analyzed by recent techniques. Besides the AT-specific fluorochromes, GC-specific fluorescent antibiotics are now available for chromosomal analysis. Chromosomal bands represent large accumulation of DNA sequences with similar AT:GC ratio. These uniform differences from the mean AT:GC ratio in the bands can be explained only by at least partial repetition of short DNA sequences in these regions. By comparison of various staining techniques more information also on the constitutive heterochromatin in man becomes available. The human NOR region exhibits a complex organization when studied by various base-specific fluorochromes and silver staining. The DNA-specific fluorochromes are also useful tools in cytophotometric DNA measurements.


Analysis of the Gc polymorphism in human populations by isoelectrofocusing on polyacrylamide gels. Demonstration of subtypes of the Gc allele and of additional Gc variants. For the study of the group-specific component (Gc) system, serum samples were examined by polyacrylamide gel electrophoresis and by a newly developed immunofixation isoelectrofocusing procedure. Thereby, a greater extent of polymorphic variation was revealed than was known previously. The allele Gc1 could be subdivided into the alleles Gc1F and Gc1S. The distribution of Gc1 subtypes was very different in three populations (Pygmies, Amerindians, and Pyreneans) examined. New variants of the Gc1 and Gc2 genes were also described in the Amerindians and in the Pygmy population, respectively.


Adjuvant and immunostimulating activities (in the absence of freund's incomplete adjuvant) of chemically modified low molecular weight mycobacterial peptidoglycans. Relatively low molecular weight peptidoglycan fragments extracted from two strains of Mycobacterium tuberculosis var. hominis were chemically coupled with lauric acid. The fatty acid conjugates were compared with the native substances with respect to some immunopotentiating activities. In vitro, the mitogenic effect on murine spleen lymphocytes was significantly enhanced following conjugation. One of the lauric acid conjugates stimulated, upon intravenous administration in mice, the formation of antibody-producing cells in the spleen, while the native substance was devoid of such activity. In adjuvanticity tests performed in the guinea pig in the absence of mineral oil, the fatty acid conjugates generally exerted a higher adjuvant effect on antibody production or on delayed type hypersensitivity reactions than did the native preparations.


High dose inhibition of the tryptophan peptide induced encephalitogenicity. In a previous publication (Immunol. Comm. 3:219, 1974) a hypothesis was proposed that the high dose inhibition of encephalitogenicity in giunea pigs seen with the tryptophan peptide, phe ser trp gly ala glu gly gln arg, and not observed with the whole myelin basic protein which contains this sequence, was the result of competitive inhibition by non-encephalitogenic fragments of the tryptophan peptide produced in vivo possibly by exopeptidases. I present data which disagrees with this hypothesis.


A new protein: macromolecular insoluble could globulin (MICG) derived from mouse thymocytes. A novel protein synthesized and isolated from mouse thymocytes is described. The protein can be separated from radiolabelled cell lysates by taking advantage of its insolubility in the cold. It is a macromolecule with a molecular weight of 225,000 daltons, migrates as a beta-globulin on electrophoresis and has unique antigenic determinents as shown by the production of a monospecific antiserum.


Rat lymphocyte differentiation antigens detected by rabbit antiserum to thymocytes and leukaemic cells. The lymphocyte differentiation antigens detected by rabbit antisera to rat thymocytes and leukaemic cells have been investigated. Thy-1 was the only such antigen detected by the anti-thymocyte serum, which was predominately directed to two xenoantigenic determinants rather than the Thy-1.1 determinant on this molecule. Two additional antigens were recognized by the anti-leukaemia serum. One of these was found on thymocytes and peripheral lymphocytes, but only on 1 per cent of bone marrow cells. The other was found not only on lymphocytes, but also on the nucleated myeloid and erythroid cells of bone marrow.


T- and B-cell surface markers on rabbit lymphocytes. Rabbit thymus, appendix, blood and lymph nodes were characterized using immunological tests: rosette-forming ability with homologous or heterologous red blood cells, surface immunoglobulins, analytical cell-electrophoresis, cell refringency and in vitro mitogen responsiveness. The experiments were conducted on normal rabbits and cyclophosphamide-treated rabbits. Moreover, we tried to separate slow and high mobility cells by free-flow electrophoresis into fractions. These experiments suggest that rabbit lymphocyte behaviour is exceptional among mammals in that the majority of their lymphocytes appeared as B cells and that, in comparison with mouse and man, the proportions of these cells are inversed.


Prevention of experimental allergic encephalitis in guinea-pigs with spinal cord protein: optimum pretreatment schedules and appraisal of plausible mechanisms. Pretreatment of Hartley guinea-pigs with three injections of 100 microgram of the purified bovine spinal cord protein, (SCP), protected tham from clinical experimental allergic encephalitis (EAE) when they were subsequently challenged with 50 microgram of purified bovine myelin basic protein (MyBP) in Freund's complete adjuvant (FCA). The length of the optimum pretreated schedule was found to be 1 week and the animals were fully protected for 2 weeks. Protection declined thereafter so that by 4 weeks following pretreatment only 40% of the animals were protected. The protected state could be restored several weeks after it had lapsed by repeating the treatment with SCP. Immature animals did not respond well to pretreatment with SCP. Evidence was presented to indicate that specific, non-specific immune suppression or anti-SCP IgG were not involved to a significant extent in protection. SCP-treated guinea-pigs regularly displayed delayed skin hypersensitivity to MyBP after sensitization but there was no correlation between the degree of cutaneous reactivity and protection from disease. Because SCP is localized in the nerve axon, the speculation was advanced that anti-SCP blocking factors might be responsible for preserving axonal function in SCP-treated animals sensitized with MyBP.


Stimulation and inhibition of macrophages prior to immunization with a hapten-carrier conjugate. Delayed hypersensitivity reactions and antibody-mediated reactions to the carrier and to the hapten have been studied in animals treated with BCG or/and trypan blue before immunization with a hapten-carrier complex. The results show that it is possible to induce, with BCG, at the beginning of the response, an antigenic competition between hapten and carrier moieties of the antigen molecule. Macrophages which modulate the response to the hapten-carrier complex by acting both on T cells involved in cellular immunity and those involved in antibody synthesis, appear to be the target of antigenic competition.


Solid-phase radioimmunoassay method for determination of Escherichia coli enterotoxin. The development of a solid-phase radioimmunoassay procedure for the determination of Escherichia coli enterotoxin(s) is described. Radioiodinated E. coli enterotoxin with about three radioiodine atoms per toxin molecule is, by the criterion of electrophoresis, identical to the unlabeled toxin. Goat anti-E. coli-enterotoxin antibody was coupled to polystyrene tubes and served as a solified toxin binder in the reported procedure. Various conditions necessary for the optimization and standardization of the solid-phase method were established. With the help of this technique it was possible to determine E. coli enterotoxin released from a porcine E. coli strain into culture medium.


Induction of interferon synthesis in mice by fractions from Nocardia. Three fractions of Nocardia, Nocardia water-soluble mitogen (NWSM), Nocardia water-soluble mitogen pellet (NWSMP), and the cell wall peptidoglycan, which are mitogenic for B lymphocytes, were able to induce circulating interferon in mice, NWSMP and NWSM being the most active. The peak of interferon appeared about 2 h after injection. The interferon induced by NSWMP and NWSM was acid stable and antigenically related to viral interferon, as shown by neutralization with antibodies directed against Newcastle disease virus-induced interferon.


Antigenic relationship between the common antigen (OEP) of Pseudomonas aeruginosa and Vibrio cholerae. Antibodies were found by the OEP-passive hemagglutination test to cross-react with the common antigen (OEP) of Pseudomonas aeruginosa in sera of rabbits immunized with two serotype (Inaba and Ogawa) strains of Vibrio cholerae. The titer in the OEP-passive hemagglutination reaction rose later than did the agglutinin titer and reached a peak of 640 to 1,280. The titers of OEP antibody formation in rabbits immunized with V. cholerae were almost the same as that of P. aeruginosa. The common antigen of P. aeruginosa was confirmed to exist serologically in both strains of V. cholerae as determined by the indirect fluorescent antibody test and the agar gel precipitin test. Passive immunization with the V. cholerae immune rabbit serum significantly protected mice against P. aeruginosa infection. Purified antibodies cross-reacting with the OEP of P. aeruginosa derived from the V. cholerae immune rabbit sera by OEP-coupled affinity chromatography protected mice against P. aeruginosa infection as compared with the control group, which was injected with 100 microgram of immunoglobin G not containing OEP antibody. The purified antibodies (2.5 microgram per mouse) protected animals challenged with approximately 10,000 50% lethal doses in the control group. Consequently, the common antigen (OEP) of P. aeruginosa proved to be a common antigen of V. cholerae both serologically and in possessing infection protective properties.


Granuloma formation by synthetic bacterial cell wall fragment: muramyl dipeptide. A synthetic muramyl dipeptide, N-acetylmuramyl-L-alanyl-D-isoglutamine, which possesses the same structure as that of a part of the peptidoglycan monomer of wax D of tubercle bacilli or bacterial cell walls was found to induce, when injected in water-in-oil emulsion, massive granulomas often accompanying abscesses in the site of injection and draining lymph nodes of guinea pigs and rats. The granulomas were composed mainly of epithelioid cells 2 weeks after injection and were indistinguishable from those induced by tubercle bacilli. The granulomas induced in rats were less mature than those induced in guinea pigs. Allergic reaction appeared to play no important role in the development of the muarmyl dipeptide-induced granuloma.


Insulin and the dextran reaction in the rat. Insulin potentiated the dextran anaphylactoid reaction in untreated rats and in rats pretreated with a protein synthesis inhibitor or with a lymphopenic agent. Incubation of rat lymphatic tissue with insulin failed to produce a factor potentiating the histamine-releasing action of dextran when this was studied in both in vivo and in vitro tests. It thus seems that insulin is exerting its potentiating effect on the dextran response through processes not involving synthesis of new protein or interaction with lymphocytes.


The inhibition of histamine release by antiallergic drugs. Three new antiallergic drugs, Doxantrazole, PRD-92 and N5', as well as disodium cromoglycate, inhibited the IgE-mediated PCA reaction in the rat triggered by the homologous antigen, but did not have an antagonistic effect on histamine itself. Moreover, all the drugs examined caused in vitro inhibition of antigen-mediated histamine release from peritoneal mast cells and chopped lung tissue of sensitized rats producing IgE antibodies. Doxantrazole had a synergistic effect on the inhibition of histamine release by isoproterenol, whereas the other drugs were devoid of this capacity. PRD-92 and N5' inhibited the ionophore A23,187 induced histamine release, but did not have any effect on the D2O-enhanced histamine release which was triggered by antigen.


Effect of a low protein diet on the capacity of mice to express a type I hypersensitivity response. The capacity of Balb/c mice maintained since weaning on a 4% protein diet to express in vivo immediate (type 1) hypersensitivity reactions was compared to that of age-matched animals maintained on a normal (18%) protein diet. The deprived mice had a lower total cellular population and lower mast cell numbers in their peritoneal cavity. However, the mean cellular histamine content was comparable between the two groups. The skin responses of malnourished mice to the histamine liberator 48/80 were depressed but both groups reacted equally to passive cutaneous anaphylaxis when sensitized with serum from immunized mice. These studies indicate that protein deprivation has not inhibited the capacity to synthesize physiologically active amines or to impair the biochemical pathway of amine release from mast cells.


Inhibition of allergic reactions by cromoglycate and by a new anti-allergy drug U-42,585E. I. Activity in rats. A new inhibitor of IgE-mediated allergic reactions has been studied in rats. This new inhibitor is chemically different from cromoglycate and shows qualitative and quantitative advantages over it. It is 2,500 times more active in rats than disodium cromoglycate and shows oral activity. This new inhibitor possesses no bronchodilator or end-organ antagonism activity and does not raise intracellular cAMP levels but as cromoglycate acts by inhibition of mediator release. Multiple high (25 mg/kg) doses in rats lead to tachyphylaxis. This effect is reversed both by decreasing the first dose and increasing the time between doses. Over a 5- to 10-fold concentration range, this inhibitor in mast cells can be stimulatory or inhibitory to 45Ca++ flux into cells. This ability to inhibit ionophore-induced 45Ca++ flux correlates well with inhibition of histamine release.


IgE-induced changes in human basophil cyclic AMP levels. We studied a unique patient with 77% basophils, not different from normal by a number of criteria, in order to measure the changes in cyclic AMP level associated with IgE-mediated histamine release. In accordance with previous hypothesis and circumstantial evidence, anti-IgE challenge led to a significant fall in the cyclic AMP level which preceded histamine release.


Possible role of IgG4 in discordant correlations between intracutaneous skin tests and RAST. Comparison of intracutaneous skin tests and RAST in 2 groups of patients, one consisting of 16 individuals having multiple allergies to pollen, mold and animal dander and the other of 10 patients allergic to mold only, revealed that skin tests were more sensitive than RAST. Skin reactions of 3+ to 4+ were frequently associated with negative RAST results. This was observed more so in mold-allergic patients. In a few cases (6%) RAST showed 2+ reactions while the skin tests were negative. Leukocytes from allergic individuals who had positive skin tests and negative RAST released a significant amount of histamine upon challenge with specific allergen as well as antihuman IgG4, suggesting that this immunoglobulin is a functional component of some immediate hypersensitivity reactions.


Unique and common tumor-specific transplantation antigens of chemically induced mouse sarcomas. Methylcholanthrene-induced sarcomas of BALB/c mice express unique tumor specific transplantation antigens (TSTA), and weaker, common TSTA. Antigens of endogenous murine leukemia virus (MuLV) expressed by some of these tumors cannot completely account for these common TSTA. However, MuLV antigens on the immunizing tumor appear to increase the immunogenicity of the common TSTA.


Production of rabbit antibodies against the viral capsid antigen (VCA) of the Epstein-Barr virus (EBV). Rabbits were immunized with nuclear or cytoplasmic extracts of the Epstein-Barr virus (EBV)-producing marmoset cell line B95-8. Following extensive absorption with human EBV-negative cells (HeP-2, Ramos and BJAB), sera were obtained that no longer reacted with cellular or serum proteins of human origin, but gave a single precipitin band with extracts of the human EBV-producing line, P3HR-1. Immunofluorescence tests performed with appropriate parallel human serum controls showed that the rabbit serum contained no activity against EBNA + EA - VCA - Raji cells, or against P3HR-1 virus superinfected, cytosine arabinoside-treated Raji cells that contained EBNA and EA, but not VCA. The sera gave a brilliant indirect immunofluorescence reaction with the virus-producing (EBNA+ EA+ VCA+) P3HR-1 lines. Two-color fluorescence tests, performed with a direct TRITC-labelled VCA conjugate and indirect FITC-staining with the rabbit serum, showed that the same cells reacted in both red and green fluorescence, confirming that the rabbit serum was directed specifically against some antigen formed in the virus-producer cells. Since the synthesis of the relevant antigen was prevented by cytosine arabinoside it cannot be EA and must be a late antigen. The morphology and localization of the antigen support the conclusion that the antigen is VCA or some part of the VCA complex.


Detection of circulating tumor antigens in mice carrying a highly metastatic pulmonary squamous--cell carcinoma. Recently we reported that the highly metastatic MSC-10 (mouse squamous carcinoma) is incapable of inducing transplantation immunity. Studies reported here were undertaken to determine whether or not the tumor is devoid of tumor-associated antigen. We found that sera from MSC-10 tumor-bearing mice contain soluble protein antigens which react with rabbit antisera made against the MSC-10 tumor, as demonstrated by immuno-diffusion. Such proteins were not detected in the sera of normal mice or mice bearing fibrosarcomas. A close temporal relationship was demonstrated between the appearance of circulating antigens and the occurrence of lung metastases. Protein components serologically indistinguishable from the circulating antigens were isolated from tumor cells. The molecular weight of these proteins is between 30,000 and 100,000 daltons. Studies with antisera to mouse leukemia virus showed that hte MSC-10 tumor antigens are not viral proteins. The lack of immunogenicity of this tumor for syngeneic hosts as well as its high metastatic activity may be due to the early appearance of soluble antigens in the circulation.


Intercellular junctions in the human developing preovulatory follicle and corpus luteum. Using the lanthanum tracer and freeze-fracture techniques, the cell contacts of human ovarian cells were examined. The cell contacts between theca interna cells are similar to those seen between luteal cells are composed of septate-like cell contacts and gap junctions. On the other hand granulosa cells are connected by desmosomes and gap junctions. Thus, at least from the viewpoint of cell junctions, it is suggested that the main precursory cells of the luteal cells are the theca interna cells, although there is a general agreement that the luteal cells are derived from the granulosa cells.


Histochemistry of B663 pigmentation: ceroid-like pigmentation in macrophages. Histochemical studies were made of pigmented cutaneous lesions from three cases of lepromatous leprosy treated with B663 to determine the nature and histogenesis of the brown pigmentation which develops as a side effect of the drug. One case of DDS-treated leprosy and four cases of untreated leprosy were also investigated histochemically as controls. The brown pigmentation of the skin is due to deposition of a ceroid-like substance in the macrophages, which is a yellowish-brown, acid-fast lipid pigment. It is insoluble in fat solvents and accepts lipid dyes even after lipid extraction by fat solvents. The macrophages in the B663-treated leprosy contain more neutral fat and less phospholipid than the untreated lepromatous leprosy tissues. Ceroid in the macrophages probably originated from unsaturated fatty acids of the leprosy bacilli through oxidation or their binding with the drug. Crystals of the drug were not found in the macrophages in this series, even on the tissues embedded in carbowax or frozen sections.


Transrectal cytological aspiration biopsy in prostatic disease. Of 761 fine-needle biopsy specimens obtained from the prostate by the method of Franzén, 303 originated from patients in whom either a histological diagnosis had been obtained or cancer had been confirmed by increased levels of prostatic phosphatases. Among palpable cancers, 83% were diagnosed by this method, and none of them gave falsely negative results. Among the cancers with benign palpatory findings and normal phosphatase levels, cytological examination did not show cancer in any of the cases. It is concluded that the method is unsuited for screening of proctatic cancer, but its use is indicated for the confirmation of the diagnosis in cases with abnormal palpatory findings, and when the diagnosis seems likely from the results of biochemical analysis and/or radiographic studies. On these indications, repeated aspiration should be done if the first biopsy specimen does not reveal cancer.


Characteristics of the Fc receptor induced by herpes simplex virus. Studies of the Fc receptor induced by herpes simplex virus using the 7S EA rosette assay reveal that (i) the percentage of cells showing receptor activity is multiplicity-dependent; (ii) the percentage of cells rosetting is independent is independent of the cell cycle phase of the host cell; (iii) RNA and viral protein synthesis, and probably glycosidation, are required for the induction of the receptor but not viral DNA synthesis; and (iv) the receptor is neuraminidase-resistant but trypsin-sensitive.


[Complete surgical correction of congenital cardiopathies in infants]. The results of total correction of congenital heart defects in the first year of life are presented. Overall surgical mortality in these generally highly symptomatic infants was 35%. The surgical risk is mainly related to the type of defect: mortality is low (10%) in uncomplicated septal defects and in transpositions of the great vessels, higher in the coarctation syndromes (30%), and considerable in complex heart defects. The weight of the infant seems to be another factor, surgical mortality being high when the patient's weight is less than 4.5 kg. Indications for palliative operation or total repair are discussed on the basis of the presented results.


Selective arterial infusion of vasoconstrictors for control of traumatic splenic hemorrhage. The spleen of each of 20 mongrel dogs was traumatized by creating a wedge laceration, and selective arterial infusion was then performed with vasopressin in four dogs, pituitrin in eight, and epinephrine in eight. Infusion therapy with vasoconstrictors controlled the splenic hemorrhage in 19 of 20 dogs. The 15 dogs surviving the experimental period of four to eight weeks showed well-healed scars at the sites of lacerations. Three dogs had small contracted spleens as a result of massive infarction. The results of this experiment indicate that splenic hemorrhage in the experimental dogs can be controlled by selective infusion of pituitrin or vasopressin, but is more effectively controlled with epinephrine.


Cytochemistry of the skin of patients with mucopolysaccharidoses. The distribution of complex carbohydrates has been investigated at the light and electron microscope levels in sweat glands of normal subjects and patients with Hurler's or Hunter's disease. Normal sweat glands examined with a battery of light microscopic histochemical methods revealed sulphated complex carbohydrate in secretory granules of the dark cells. These granules lacked affinity for dialysed iron (DI) at the light and electron microscope levels. The DI method demonstrated acid complex carbohydrates ultrastructurally on the surface of the intercellular canaliculi and central lumen in normal sweat glands. DI-reactive acidic material, presumably of mucopolysaccharide nature, surrounded and extended between collagen bundles in the stroma of normal skin, but was absent from the band which ensheathed the sweat gland and consisted of individual rather than bundled collagen fibrils. DI-reactive mucopolysaccharide lined and partially filled vacuoles of dark cells showing a laminar distribution in vacuoles of clear cells in sweat glands of a Hunter patient. The DI method also visualized mucopolysaccharide distributed throughout vacuoles in fibroblasts of this patient. DI-reactive acid material covered the luminal surface of the sweat gland, coated collagen bundles in the stroma and spared the periglandular collagenous sheath in skin from Hurler and Hunter patients as in that from normal controls. Acid phosphatase was localized ultrastructually in vacuoles and nearby cytoplasm and on plasmalemmae of clear cells, dark cells and myoepithelial cells of sweat glands from Hurler and Hunter patients. Vacuoles of dermal fibroblasts and Schwann cells in these specimens also exhibited strong acid phosphatase activity.


Changes in elemental composition of human chromosomes during a G-banding (ASG) and a C-banding (BSG) procedure. Human chromosomes fixed in methanol-acetic acid have been examined by X-ray microanalysis, before, during and after a G-banding and a C-banding procedure. Phosphorus (representing mainly DNA), sulphur and calcium are the most prominent elements in untreated chromosomes. In the G-banding procedure, the calcium is lost during 2 x SSC treatment. In the C-banding procedure, calcium is lost in the preliminary HCl treatment. During the following barium hydroxide treatment a large amount of barium becomes attached to the chromosomes, but is lost again during the subsequent 2 x SSC treatment. In both banding techniques Giemsa staining produces large peaks for sulphur (thiazine dyes) and bromine (eosin), showing that both types of dyes are involved in the staining. Reduction in the phosphorus peak during these procedures may be partly due to extraction of DNA and other chromosomal components, but could also be due to absorption of phosphorus X-rays by heavy elements (barium and bromine).


Cobalt thiocyanate as a stain for basic proteins and other organic bases on thin sections. Thin sections in mouse mast cells and thymic cells are stained with cobalt thiocyanate a compound known to form insoluble complexes with organic bases. Chromatin, nucleolus, ribosomes and mast cell granules are contrasted. Different blockade reactions and enzymatic digestions indicate the staining corresponds to the basic protein amino-groups. The silver methenamine reaction stains the same cellular structures. However, the specificity control reactions show the staining mainly corresponds to protein sulphydryle groups and in a lesser extent to aldehyde and polyanions.


A microprobe analysis of Gomori-positive glial cells in the rat arcuate nucleus. Astrocytes and microglial cells in the arcuate nucleus of the rat hypothalamus contain lipofuscin-like granules which react with chrome alum gallocyanin and exhibit endogenous peroxidase activity. These granules were assessed with energy dispersive X-ray microanalysis and compared to neuronal dense bodies and glial cytoplasm. The granules are distinguished by a consistent content of sulphur and a frequent presence of calcium. The localization of other elements such as iron, copper, potassium and chlorine is impaired by methodical difficulties. The sulphur content as well as the endogenous peroxidase activity is interpreted as indicating a special variant of lipofuscin. The presence of calcium is discussed with respect to recent concepts of glia as a regulator of the ionic environment of the CNS.


[Azoindoxyl methods for the investigation of hydrolases. III. Histochemical studies of beta-D-N-acetylglucosaminidase (author's transl)]. The suitability of various azoindoxyl procedures for the light microscopical demonstration of beta-N-acetylglucosaminidase is described. The incubation media tried consist of 0.5 mg N-Acetyl-(5-bromindol-3-yl)-beta-D-glucosaminide (5-Br-3-indolyl-beta-D-N-acetylglucosaminide; 1 mg dissolved in 0.05 ml N,N-dimethylformamide) in 1 ml 0.1 M citric acid phosphate buffer, pH 4.5 or 5. 0.02 ml hexazotized p-rosaniline or new fuchsine/ml or tetrazotized BAXD or 0.5 mg Fast Blue B or Garnet GBC/ml were employed as a coupling reagent. Hexazotized new fuchsine yields the best results independent on the pretreatment of the tissue and the organ investigated followed by hexazonium-p-rosaniline. Compared with the azo dye method using naphthol AS-BI beta-D-N-acetyl-glucosaminide as a substrate and hexazotized p-rosaniline or new fuchsine or tetrazotized BAXD for simultaneous coupling especially the azoindoxyl technique with the new fuchsine is equvialent or superior. When the indolyl glucosaminide is used in the indigogenic, tetrazolium or metal precipitation method the results are mostly inferior with the exception of the tetrazolium reaction using BSPT. However, the main advantage of the azoindoxyl procedure is that at least the azoindoxyl dye deriving from hexazotized p-rosaniline can be osmificated and withstands treatment with organic solvents and resins. Therefore, the reaction product seems to be suitable for the electron microscopic demonstration of glucosaminidase. Among the other reaction principles this can reliably be achieved only with BSPT as a tetrazolium salt followed by osmification of its formazan. After fixation of blocks of tissue in form- or glutaraldehyde beta-D-N-acetylglucosaminidase can be localized with 5-Br-3-indoxyl-beta-D-N-acetylglucosaminide as a substrate and hexazotized new fuchsine for simultaneous coupling in the lysosomes of many rat organs.


Distribution oand pharmacological release of histamine in canine lung in vivo. We have examined the physiological effects of stored mediators released from airways by compound 48/80 aerosols in anesthetized dogs. In 13 dogs, both mast cell numbers and tissue histamine were related inversely to bronchial internal diameter (P less than 0.0001). Compound 48/80 aerosols degranulated mast cells and decreased histamine content (-29.0 +/- 10.0%; mean +/- SE) in 5-10 mm bronchi, but not in 3-4 mm bronchi or lung parenchyma. This was associated with increased plasma histamine (31.8 +/- 18.4 ng/ml), increased airflow resistance (Rrs: + 452 +/- 257%), decreased lung compliance (-28 +/- 10%), and decreased arterial blood pressure (-41 +/- 6.5%) at 2 min. The increased Rrs was reversed by beta-adrenergic agonists, indicating it was caused by bronchial smooth muscle contraction; prevented by chlorpheniramine, indicating it was caused by histamine action on H1-receptors; and augmented and prolonged by propranolol, suggesting that histamine triggered sympathetic mechanisms which modulated the effect of 48/80. This experimental approach permits the study of mechanisms in vivo which may be involved in the sequence of reactions initiated by antigen-IgE interaction. However, the latter involve not only stored mediators, but also unstored mediators, neural reflexes, and complex cellular interactions.


Propagation and histological characterization of a homotypic population of astrocytes derived from neonatal rat brain. Tissue derived from the cerebra of 3-4 day old rats was minced, trypsinized and placed in tissue culture medium. Cells attained a monolayer in twelve to fourteen days when plated at a density of 10(2) cells/cm2. Selected culturing procedures designed to inhibit neuronal growth while encouraging glial growth yielded monolayers which consisted almost exclusively of astrocytes. Cells were identified using established morphological criteria for brain cells in culture. These observations were supplemented using certain histological and histochemical techniques frequently employed in investigative work for the identification of glial cells, particularly astrocytes. Results of all the above procedures indicate cell cultures which apparently consist exclusively of astrocytes. The preparation of such a homotypic population of normal, viable astroglial cells, derived from an animal universally used in investigative work on the nervous system, is perceived as an important contribution to the future study of glial characteristics and their interactions at morphological and functional levels.


Unilateral ptosis as a conversion reaction. This case report affords the dynamic understanding of a patient's conversion reaction of unilateral ptosis of the eye. The specificity of this manifestation of psychopathology is shaped by the patient's unconscious ideas about disease and the suitability of this symptom for the symbolic representation of her primary conflict.


Further studies on endo-beta-N-acetylglucosaminidase D1. The purification procedure for endo-beta-N-acetylglucosaminidase D was improved to yield an enzyme preparation which was homogeneous upon gel electrophoresis. The molecular weight of the enzyme as estimated by Sephadex G-200 column chromatography was 280,000, while SDS-gel electrophoresis after reduction with 2-mercaptoethanol gave a value of 150,000. The purified enzyme did not show any chitinase, hyaluronidase or lysozyme activity. In the presence of exoglycosidases removing peripheral sugars, the endoglycosidase acted on serum glycoproteins such as transferrin and fetuin. The enzyme also hydrolyzed an oligosaccharide, (Man)5(GlcNAc)2, indicating that the peptide portion of substrates does not have much effect on susceptibility to the enzyme.


The presence of fatty acids in human alpha-fetoprotein. alpha-Fetoprotein has been prepared from human fetal tissue by procedures utilizing DEAE-Sephadex, concanavalin A-Sepharose, and isoelectric focusing. A major and a minor component with isoelectric points of 4.7 and 5.3, respectively, have been isolated and are similar to those prepared under various conditions by other investigators. The 4.7 material contains 2.4 mol of fatty acids/mol of protein, whereas the minor component is fat-free. The relative amounts of fatty acid vary somewhat with different preparations. The ranges found in three isolates were as follows: palmitic acid (8 to 11%), stearic acid (2 to 5%), oleic acid (10 to 28%), linoleic acid (7 to 15%), arachidonic acid (12 to 39%), and 4,7,10,13,16,19-docosahexaenoic acid (16 to 42%). Human fetal serum albumin contained 0.7 mol of fatty acid/mol of protein, with arachidonic acid and the docosahexaenoic acid comprising only 11.4% of the total. Removal of fatty acids by treatment with charcoal converted alpha-fetoprotein into material with an isoelectric point of pH 5.3. Addition of arachidonic acid to the lipid-free protein restored it to protein with a pH 4.7 isoelectric point, typical of the major native component. The possible role of the fatty acids in alpha-fetoprotein on the inhibition of various lymphocyte functions is projected.


Synthesis of double-stranded DNA complementary to lysozyme, ovomucoid, and ovalbumin mRNAs. Optimization for full length second strand synthesis by Escherichia coli DNA polymerase I. Sequential reverse transcriptase, DNA polymerase, and S1 nuclease reactions can be employed to synthesize double-stranded DNA representing messenger RNA. Using reverse transcriptase products made from partially purified lysozyme, ovomucoid, and ovalbumin messengers from hen oviduct, we have characterized the Escherichia coli DNA polymerase I reaction. We have optimized for a high yield of full length second strands under conditions which require only a small amount of mRNA. The effects of several parameters (time, enzyme levels, salt concentration, monovalent cation, and temperature) on the length of products synthesized by DNA polymerase I have been investigated. Each has a significant influence on the proportion of products which are full length. Under our conditions the three reactions are efficient in synthesizing full length duplex DNA from partially purified mRNA fractions or from total poly(A)-containing RNA.


Efficacy of direct Gram stain in differentiating staphylococci from streptococci in blood cultures positive for gram-positive cocci. A preponderance of clusters seen on direct Gram stain of blood cultures positive for gram-positive cocci was 98% sensitive and 100% specific for identification of staphylococcal species or of Peptococcus. A preponderance of chains, pairs, or both was 100% sensitive and 98% specific for identifying streptococci. Further presumptive identification of either staphylococci or streptococci based on microscopic morphology was unreliable. The direct Gram stain is highly reliable for differentiating staphylococci from streptococci and should be of considerable value to clinicians selecting initial antimicrobial therapy.


Contamination of primary embryonic bovine kidney cell cultures with parainfluenza type 2 simian virus 5 and infectious bovine rhinotracheitis virus. Two different viruses were isolated from bovine embryonic cell cultures after two subcultures from the primary cells. One virus was identified as parainfluenza type 2 simian virus 5 (SV-5), and the other was identified as infectious bovine rhinotracheitis virus. Six months later, stock cultures of pig kidney (PK-15) cells were found to be contaminated with SV-5 virus. We believe that the source of the SV-5 virus in the bovine cells was a cross-contamination from monkey kidneys during preparation of the cell cultures. The infectious bovine rhinotracheitis contamination was probably of endogenous origin. The bovine embryonic cell cultures were the probable source of contamination of the PK-15 cells with SV-5 virus.


Staining clinical specimens for acid-fast bacilli by means of a mechanical conveyor system. A Cyto-Tek staining machine designed for Papanicolau staining of cervical smears has been adapted for auramine staining of smears of sputum and other clinical specimens for acid-fast bacilli. The results compared favourably with those obtained by a manual method of staining. Contamination of negative smears by acid-fast bacilli from positive smears was not found. Since this study the machine has continued to be in routine use with a considerable saving in labour.


Organization of the mossy fiber system of the rat studied in extended hippocampi. II. Experimental analysis of fiber distribution with silver impregnation methods. The mossy fibers, a major intrinsic hippocampal pathway connecting the dentate granule cells with the pyramidal cells in CA4 and CA3, have been reexamined in rats using mainly Fink-Heimer silver impregnation methods for demonstration of degenerating axons. By extending isolated hippocampi and cutting sections normal to the long axis, simple two-dimensional reconstructions of both the lesions and the resultant degeneration could be made. In the hilus, the zone with the greatest concentration of degenerating boutons was found between the lesioned granule cells and the CA3 pyramidal cells abuting on the hilus; outside this zone the concentration declines rather rapidly. Degenerating boutons were also observed in low concentration up to 200-300 microgram septal and temporal to the lesion. The mossy fibers in CA3 nearest the hilus have an intrapyramidal course and display a lamellar organization with fibers from the granule cells of the medial blade lying deep to those from the dentate crest. These in turn lie deep to those from the graule cells of the lateral blade. A mediolateral difference in the projection of the graule cells on the CA3 pyramidal cells was discovered: fibers from the medial granule cells descend about 600 micrometer in the temporal direction, whereas fibers from the lateral granule cells descend about 1,200 micrometer. This causes a divergence of the fibers from one single level, especially of the part of the fibers, being farthest away from the hilus. The degree of descent of the fibers from each mediolateral position of the granule cells was constant at all septotemporal levels examined.


Alterations of clotting factors and platelets during hemofiltration. The influence of hemofiltration on the number of platelets and on coagulation factors was investigated in patients with chronic renal insufficiency. These investigations were done on 12 patients during 22 treatments with hemofiltration. Blood samples were taken before hemofiltration, 10, 30 and 120 minutes after the beginning of the treatment and at the end of hemofiltration. In comparison to the original values we found a loss of platelets, a small decrease in the concentration of fibrinogen and a small increase in the fibrin monomer complex, plasminogen, antithrombin III, alpha1-antitrypsin and in alpha2-macroglobulin. The thrombin time, the partial thromboplastin time and Quick's test showed that the blood of these patients contained sufficient hepatin. Use of fibrin plates (Astrup) showed no signs of fibrinolytic activity. Compared to the results, which were obtained some years ago during hemodialysis, we found a smaller extent of alterations of blood coagulation factors and number of platelets.


Improving slide illustrated presentations. Slide illustrated presentations can be improved with dual image projection. A dissolve control unit can be used to enhance the presentation of sequence material and also can be incorporated into the dual image system of the slide delivery.


The effect of filmed modeling on cooperative play in disadvantaged preschoolers. In a series of three studies wer investigated the effect of filmed modeling on cooperative play in disadvantaged preschoolers. These studies were conducted both in a natural (classroom) setting and in a laboratory setting. We were unable to demonstrate a reliable modeling effect with these preschoolers even when an extensive descriptive verbal component was added to the modeling film. In a fourth study we showed that the identical treatment did lead to modeling in middleclass preschoolers but that the content of the extensive verbal component was not related to the effect. These results are discussed in terms of appropriate control procedures for this type of study, the role of an audio component in modeling films, and the implications of the lack of filmed modeling effects for the teaching of disadvantaged preschoolers.


Effect of pre-exposure on acute toxicity of organophosphorus insecticides to white mice. LD50 and in vitro ChE I50 values of Chlorpyrifos, Leptophos, Phosfolan, and Stirophos against white mice showed that the formulated insecticides were higher in their mammalian toxicity than the corresponding technical materials. Pretreatment of mice with a sublethal dose of Phosfolan potentiated the toxicity of post-treatment with formulated Stirophos, Phosfolan, or Chlorpyrifos, but antagonized the toxicity of post-treatment with Leptophos. On the other hand, pretreatment with sublethal doses of Leptophos resulted in potentiation of Stirophos or Phosfolan, but decreased the toxicity of Chlorpyrifos or Leptophos. Pretreatment of mice by sublethal dose of Phosfolan synergized the in vivo inhibitory power of post-treatment by Phosfolan, Chlorpyrifos or Leptophos against brain and Plasma ChE. On the other hand pretreatment with sublethal doses of Leptophos antagonized the inhibitory power of post-treatment with either Chlorpyrifos, Leptophos or Stirophos against mice brain-ChE.


The in vitro metabolism of lindane by an enzyme preparation from chicken liver. The soluble fraction (105,000 x g, 30 min) of chicken liver homogenates contained an enzyme(s), probably a dehydrochlorinase(s), which metabolized lindane in vitro. The reaction was glutathione dependent and took place anaerobically. The enzyme(s) also metabolized the alpha- and delta-isomers but not the beta-isomer. About 66% of the in vitro metabolites were soluble in petroleum ether. From the ether-soluble fraction, o-, p- amd m-dichlorobenzene, 1,2,3- and 1,2,4-trichlorobenzene, gamma-2,3,4,5,6-pentachlorocyclohex-1-ene (gamma-PCCH), 2,3- and 2,4-dichlorophenol and 2,4,6-trichlorophenol were identified by comparison of mass spectra and gas chromatographic data with those of reference compounds. Seven additional metabolites were tentatively identified from their mass spectra data. These metabolites were: chlorobenzene, 2 isomers of trichlorocyclohexene, dichlorocyclohexadienetriol, chlorophenol, trichlorocyclohexenol and trichlorocyclohexanediol. The petroleum ether-extracted aqueous phase contained a number of unidentified conjugated metabolites. It was concluded that the metabolic pathway for lindane in the chicken is quite similar to that in the pheasant.


Flow-cytometric analysis of chicken red blood cells. Flow-cytometric analysis of acriflavin-Feulgen stained chicken erythrocytes shows a complex distribution of amounts of deoxyribonucleic acid fluorescence, the profile consisting of a main peak and a right hand shoulder. This bimodal distribution, an artifact characteristically seen on analysis of flattened cells using orthogonal flow systems, results from fluorescence emission in preferred directions stemming from the combined effects of refractility and orientation of the cells. The shoulder disappears on analysis of lysed erythrocyte ghosts, also on analysis of cells in a medium whose refractive index approximates that the cells. An orientation effect for matrue erythrocytes was indicated by reanalysis of fractions after sorting on the basis of high and low fluorescence or scatter signals. Both fractions gave the original range of values on reanalysis, although some changes in shape of the profile and in the peak positions for the sorted cells were seen. Sodium dodecyl sulfate treatment of stained cells "loosened" the cells' structure, yielding lowered scatter values, and fluorescence values approaching those of the shoulder. The average fluorescence emission of the erythrocytes was lower than that of reticulocytes and lymphocytes. The values of the latter correspond closely, although coincidently, to that the erythrocyte shoulder values. Dual parameter analysis of forward light scatter, and fluorescence, which was detected at 90 degrees to the laser beam, showed the low fluorescence to be accompanied by low scatter signal, and the high fluorescence among the cells with the high scatter signal. The lowered forward scatter signal is due to a wider scattering of light from cells oriented edge-on to the detector, and loss of signal beyond the acceptance angle of the detector. These results suggest that the preferred directions for fluorescence are in the plane of the cells, and the values are dependent on the cells' orientation in the stream. These interpretations were supported by the results of analysis of partially oriented cells. The approaches used and conclusions arrived at are similar to those of Gledhill et al (16), Van Dilla et al (37), in their analysis of fluorescence of flat sperm cells although the affects in the case of the erythrocytes are less extreme.


Geographical distribution of the dermatophytes: a review. In these days of rapid transit from continent to continent, and the increasing mobility of people, agents of disease are no longer geographically restricted. Disease contracted half way across the world may become manifest in a country in which the pathogen is not normally found. Thus knowledge of the geographical distribution of pathogens becomes increasingly important when a diagnosis is being made. This is as true of ringworm fungi as of any other group of microorganisms. In the last 12 years, in the Mycological Reference Laboratory, an increasing number of exotic dermatophytes have been seen, related in part at least to the great increase in the number of non-British residents. Not all species of dermatophytes are cosmopolitan in their distribution throughout the world. While some have been recorded from every continent, others have geographically limited areas of greater or lesser extent. Surveys taken at intervals in a country may show a rise and fall in occurrence of several species as habits change, populations move and medical facilities became increasingly well-distributed. There have been few geographical surveys of ringworm fungi that have covered the world. Ajello (1960, 1974) has reviewed the individual species with regard to geographical location, while Vanbreuseghem & de Vroey (1970) attempted to estimate the relative importance of the various species in terms of numbers of isolations reported. This paper therefore reviews the world dermatophyte flora in terms of the dominant agents in the various countries, and some of the changes that have been recorded.


T cell tolerance in the chicken. I. Parameters affecting tolerance induction to human gamma-globulin in agammaglobulinemic and normal chickens. T cell-mediated delayed hypersensitivity (DH) to human gamma-globulin (HGG) can be induced in chickens by subcutaneous injection of the antigen in complete Freund's adjuvant (CFA). In the present work, it has been demonstrated that specific tolerance of the cells mediating this DH can readily be induced in both normal and bursectomized (BX) FP strain chickens by simple i.v. injection of soluble antigen, regardless of the presence of antibody production to the tolerogen. A significant degree of tolerance at the DH and helper T cell levels could be generated in BX birds by injection of as little as 0.5 mg of HGG; such a dose only induced tolerance in normal birds when it had been previously deagregated by ultracentrifugation. Regular, nondeaggregated antigen could produce tolerance in normal animals, but only at doses of greater than 5 mg. The tolerizing injection induced a primary antibody response in normal birds in all cases, but a secondary response could not be obtained in animals rendered tolerant at the T cell level. Establishment of tolerance appeared to be very rapid, and animals remained refractory to induction of DH for at least 3 weeks after the tolerizing injection. The mode in which the antigen was presented to the animals appeared to be crucial in determining whether tolerance or sensitivity would be established.


Nonimmunospecific protein-protein interactions of IgG: studies of the binding of IgG to IgG immunoadsorbents. Nonimmunospecific interactions of IgG and IgG-agarose columns were systematically studied under varying conditions. Nonimmunospecific binding to the columns was primarily due to protein-protein interactions. These nonimmunospecific protein-protein interactions of IgG were enhanced with heat-induced or chemical aggregation of IgG, low pH, low ionic strength (at pH above 4), or low temperature. Conversely, this binding was decreased with proteolytic fragmentation of IgG, high ionic strength (at pH above 4), or temperatures above 4 degrees C. Chemical modification of IgG by acetylation, formalinization, carbamylation, or reaction with 1,2-cyclohexanedione significantly decreased these interactions. These observations suggest that above pH 4, ionic interactions caused the protein-protein binding. Below pH 4, hydrophobic interactions presumably play a major role. These results permit the development of rational methodology for avoiding nonimmunospecific protein-protein interactions in immunologic procedures for detection, isolation, or quantification of rheumatoid factors and other antibodies to IgG.


Triggering of histamine release from rat mast cells by divalent antibodies against IgE-receptors. Antibodies against receptor molecules for IgE on rat basophilic leukemic (RBL) cells were prepared by immunization of a rabbit with immune precipitates composed of IgE-receptor complexes and anti-IgE. Antibodies against cell surface components were specifically purified by using RBL cells and rendered specific for mast cells by appropriate absorption. The major antibodies in the final preparation (anti-RBL) were directed against receptor molecules. It was found that the F(ab')2 fragments of anti-RBL induced histamine release from rat mast cells and caused immediate skin reactions in normal rats. These reactions by anti-RBL or its F(ab')2 fragments were inhibited if the receptors on mast cells had been saturated with IgE. The Fab' fragments of anti-RBL could bind with receptors on RBL cells and blocked passive sensitization of mast cells with IgE antibodies, but failed to induce skin reactions and histamine release from normal mast cells. Sensitization of normal rat skin with the Fab' fragment followed by an i.v. injection of anti-rabbit IgG induced skin reactions. The results indicated that bridging of receptor molecules by divalent anti-receptor antibody triggered mast cells for histamine release.


Complement-dependent stimulation of normal lymphocytes by immune complexes. Normal rabbit lymphocytes were stimulated to proliferate in vitro by antibody-antigen complexes. Stimulation was dependent upon C activity. Heat-inactivation or zymosan-treatment of the serum used in culture caused a 75 to 100% loss of responsiveness to the complexes. Serum-free culture or cultures with less than 1% serum supported only low levels of stimulation, but responsiveness reappeared proportionally with increased serum concentration. The low level dose-dependent responses seen in the absence of active C may have been due to C carried over with the cells or to stimulation independent of C. Aggregated rabbit gamma-globulin tested over a broad dose range failed to stimulate normal lymphocytes more than minimally whether or not C was present. Stimulation with immune complexes was sustained by C4-deficient guinea pig serum, indicating participation of the alternative C pathway. Normal rabbit lymphocytes from peripheral blood, bone marrow, spleen, and lymph node proliferated in response to rabbit antibody-antigen complexes. Normal thymocytes were consistently unresponsive to the complexes.


Structural and genetic studies on chicken 7S immunoglobulin allotypes. IV. The presence of an unexpected chicken immunoglobulin heavy chain allotype: subclass or pseudoallele? Low concentrations of allotypic specificity CS-1.1 were detected in the sera of two inbred chicken lines [University of California, Davis (UCD) 7 and Regional Poultry Research Laboratory 15I4] previously reported to lack this specificity. The CS-1.1 alloantigen in 15I4 chickens has the same specificity as the major allotype in a line of chickens (UCD 2) in which it was initially defined. In 15I4 chickens, CS-1.1 allotype is present on a population of molecules distinct from those which carry the major allotype; thus a second 7S Ig H chain locus, CS-2, is proposed. The concentration of CS-1.1-bearing molecules determined by two different methods was 7 microgram/ml and 230 microgram/ml in 15I4, whereas UCD 2 chickens had 4 mg/ml of CS-1.1 molecules. The levels of CS-1.1 inhibitory activity in 15I4 birds remained relatively constant over a 30-day period. The presence of two 7S Ig populations in 15I4 chickens may be interpreted as evidence either for 7S Ig subclasses with shared allotypes or for a pseudoallelic organization of genes controlling expression of 7S Ig H chains. The results were consistent with the presence of redundant C region genes, differing in allotypes, whose expression is under the control of an as yet undefined regulatory mechanism.


Expression of murine Ia antigens during embryonic development. An immunochemical analysis of the kinetics of appearance of Ia antigens during embryonic development was performed. Ia antigens first appear on the surface of embryonic cells 11 days postconception and their expression between days 11 and 16 of gestation is confined to the fetal liver. Ia antigen synthesis by fetal liver cells is detectable at day 14. Ia seems to precede Ig as a surface marker of embryonic liver cells, since Ig cannot be detected until day 16 of gestation. H-2 antigens may be immunoprecipitated from day 10 whole embryo cells. F9 primitive teratocarcinoma cells are Ia negative and H-2 negative.


Delineation of spontaneous erythrocyte autoantibody responses of NZB and other strains of mice. Four anti-erythrocyte autoantibody responses (anti-X, anti-HB, anti-HOL, and anti-I) that occur spontaneously in mice have been characterized with regard to antigenic specificities, predominant immunoglobulin class, and pathogenetic importance. Each autoantibody response exhibits specificity for an independent erythrocyte membrane autoantigen (X, HB, HOL, or I) or a soluble analogue (SEA-X or SEA-HB) present in the plasma. The anti-X response, unique to NZB mice, is directed to a normally exposed murine erythrocyte autoantigen, whereas the anti-HB response is directed to a cryptic erythrocyte autoantigen exposed by limited enzymatic cleavage of the membrane. The anti-I response also is directed to a cryptic but distinct autoantigen, and anti-HOL autoantibodies react with an erythrocyte autoantigen located at the cytoplasmic surface of the membrane. Analysis of the predominant immunoglobulin class of each of the autoantibodies has demonstrated that anti-HB and anti-I antibodies are predominantly of IgM class, whereas anti-X and anti-HOL antibodies are IgG immunoblobulins. Only anti-X and anti-HB autoantibodies are recovered from Coombs' positive erythrocytes from NZB mice and erythrocytes with surface C3 are detected only in NZB mice greater than 9 months of age. These data suggest that only the anti-X and anti-HB responses are pathogenetically implicated in the autoimmune hemolytic anemia of NZB mice.


Suppression of growth of guinea pig line-10 hepatocarcinoma. I. Effect of simultaneous administration of tumor cells and antibodies from normal rabbits. Suppression of growth of the line-10 hepatocarcinoma in strain-2 guinea pigs occurred when line-10 cells were injected intradermally together with sera or immunoglobulins derived from normal rabbits. A significant number of animals were resistant to subsequent rechallenge with tumor cells. This immunity was specific, depended on contact of immunoglobulins with tumor cells and on the concentration of immunoglobulins. Repeated injections acted as potent vaccines and resulted in the development of immunity in 84.6% of recipients. Fc receptors were not detected on line-10 cells. Antibodies reacting with line-10 cell unique antigens as well as with antigens common to line-10, line-1 and normal guinea pig spleen cells were found in NRS. Injection of line-10 cells together with rabbit immunoglobulins from which antibodies reacting with antigens derived from line-10 cells had been removed did not result in tumor suppression. The specific antigen(s) recognized by antibodies that suppressed growth of the line-10 tumor in vivo was not determined.


Tolerance induced by TNP-derivatized syngeneic erythrocytes: evidence for cooperation between hapten-specific T and hapten-specific B lymphocytes in the immune response. Tolerance to the DNP haptenic determinant was induced with a single i.v. injection of trinitrophenylated syngeneic red blood cells. The tolerant state lasted 1 month and was stable on transfer to irradiated thymectomized syngeneic recipients. Suppressor activity was found soon after injection of tolerogen but was lost before the termination of tolerance. The unresponsive state could be reversed by adding normal thymus cells to tolerant spleen cells but not by normal bone marrow cells. LPS when given with immunogen restored the normal immune response in tolerant mice. Thus the injection of TNP-MRBC induced partial immune unresponsiveness which was characterized by the induction of T cell suppressor activity and by a hapten-specific helper T cells tolerance. Finally, these studies suggest a cooperative interaction between DNP-specific T lymphocytes and DNP-specific B lymphocytes in the immune response to DNP-BGG.


Leukocyte complement: neoantigens of the membrane attack complex on the surface of human leukocytes prepared from defibrinated blood. The neoantigenic determinants (neoAg) which have been identified in the human C5b-9 membranolytic C complex were detected here by the direct fluorescent antibody technique on the surface of 27 +/- 11% of viable peripheral blood leukocytes (PBL). The cells were prepared from defibrinated blood by sedimentation on Ficoll-Hypaque. Specificity of the antisera was established by quantitative inhibition of the fluorescent staining reaction, and of agglutination of EAC1-7, by highly purified C5b-9 complex. No inhibition was observed with fresh normal human serum. The majority of the PBL with surface neoAg was found in the B lymphocyte subpopulation that failed to form rosettes with sheep erythrocytes. NeoAg on B lymphocytes was removed to differing degrees by trypsin, papain, or pepsin treatment, and by maintaining the cells at 4 degree C for 20 hr in serum-free medium. The individual components, C5, C6, C7, C8, and C9, were also detected on the surface of PBL. With differential fluorescent stains, C5 and neoAg as well as C8 and neoAg could be detected on the same cells. The results indicate that viable B lymphocytes prepared from defibrinated blood, have the components of the membrane attack complex of C on their surface. The concomitant occurrence of the neoAg indicates that these proteins are present at least in part in the form of the assembled terminal complex.


Intrinsic tritium labeling of the capsular polysaccharide antigen of Haemophilus influenzae type B. The capsular polysaccharide of Haemophilus influenzae type b was intrinsically labeled with tritium by a microculture technique with 6-3H-D-glucose and was isolated in radioantigenically pure form by a combination of selective precipitation and molecular sieve chromatography. Labeling with tritated sugar residues approached one-fourth maximum and produced a specific activity 10-fold that previously described for extrinsic labeling methods. In radioantigen-binding assays for antibody, sensitivity depended on the size of the antigen; preparations were readily made that could detect 0.01 microgram Ab/ml in serum samples of 25 microliter. Stability of the labeled antigen appears limited only by the primary radiodecomposition of tritium.


Epidermal metabolism in heredopathia atactica polyneuritiformis (Refsum's disease). The epidermal metabolic activity of a patient with a marked generalized ichthyosis associated with heredopathia atactica polyneuritiformis has been investigated. Both the in vivo labeling index and the in vitro rates of incorporation of radioactively labeled thymidine, proline, histidine and acetate were increased relative to normal indicating a high rate of epidermopoiesis. Thin-layer chromatographic analysis of 14C-acetate containing lipid extracts revealed qualitative changes compared with normal. In particular, altered proportions of radioactivities were incorporated into the triglyceride and phospholipid moieties. However, as abnormal patterns of lipogenesis are also seen in autosomal dominant ichthyosis, these changes are probably a reflection of disordered keratinization.


Localization and synthesis of alphafoetoprotein in post-implantation mouse embryos. The localization and synthesis of alphafoetoprotein (AFP) during mouse embryogenesis were studied by immunoperoxidase and by immunoprecipitation after radioactive labelling, using an antiserum prepared against AFP. AFP is first detectable in embryos on the 7th day of gestation (7th day embryos). In 7th and 8th day embryos AFP is confined to visceral (proximal) endoderm cells around the embryonic region of the egg cylinder. Visceral extra-embryonic and parietal (distal) endoderm cells do not contain AFP. By the 9th day of gestation AFP is also present in the extra-embryonic ectoderm, mesoderm and embryonic ectoderm cells around the three cavities of the embryo. These tissues do not synthesize AFP when cultured in isolation, but can adsorb AFP when it is added to the medium. On the 12th day of gestation AFP synthesis is confined to the endoderm layer of the visceral yolk sac. It is concluded that the ability to synthesize AFP is a property which is restricted to the visceral endoderm during early post-implantation development. The presence of AFP in other tissues of the embryo appears to be due to adsorption.