Combined therapy of oral cancer bleomycin and radiation: a clinical trial. A concurrent randomised controlled clinical trial to evaluate a combination of Bleomycin and radiation as against radiation only in the treatment of advanced oral cancer has been conducted at the Cancer Institute, Madras, since 1971. All T3 and T4 previously untreated oral squamous cell carcinomas with N0, N1 and N2 regional nodes, or N3 nodes confined to the submandibular region, without systemic metastases or gross infiltration of the temporal and infratemporal fossa producing total trismus, and in decent general health were eligible for the trial. Patients with gross active pulmonary tuberculosis were excluded, as were recurrent carcinomas. Age, external fungation of growth or radiological bone invasion were no bar. Randomisation was done by the sealed envelope technique. The study group received concurrent fractionated cobalt 60 teletherapy using two opposing fields and 10-15 mg of Intra-arterial or Intravenous Bleomycin. The controls received fractionated cobalt teletherapy and i.v. or i.m. distilled water on the same protocol as the Bleomycin cases. All cases were evaluated double blind 8 weeks after the end of radiation therapy, and were classified as 'favourable response' or 'failure'. The criterion of 'favourable response' was 'total clinical healing of the tumour within the volume of irradiation with no subsequent recurrence within that volume, whatever the length of follow up'. Anything else was reported as a failure. A long term follow up of 3 years is also available. 136 cases have completed the trial. The favourable response in the study group was 77% as against 20.9% in the control group. The differential response is statistically significant. The present study is the fourth in the series of combined therapeutic trials conducted in advanced oral squamous cell carcinoma since 1958. (Krishnamurthi and Shanta, 1963, 1965, 1967 and 1971). A concurrent randomised controlled clinical trial to evaluate the combination of Bleomycin and radiation as against radiation only in treatment of advanced oral cancer has been conducted at the Cancer Institute, Madras since 1971.


Japanese medicine in Manchuria: the South Manchuria Medical College. During the eleven years of its existence as Namman Igakudo, the school graduated 421 physicians; after it was upgraded to a university-level school in 1922, there were, 1,181 graduates until the college closed as a Japanese institution in 1946. There were over 500 graduates from the second-level, Igaku Semmon-bu, between 1929 and 1946. The graduated of the school became leaders of Japanese-sponsored programs in medicine and public health in Manchuria. Others returned to the home islands as practitioners; those who wished to enter academic medicine usually turned in time to Kyoto University Faculty of Medicine. The S.M.M.C. played a unique role in medical research in Japan. Further, it became one of the leading medical schools in China.


Collagenase and its natural inhibitors in relation to the rheumatoid joint. This report attempts to summarize our present knowledge of rheumatoid synovial collagenase and its natural serum inhibitors, beta1-anticollagenase and alpha2-macroglobulin, in relation to cartilage collagen resorption in the rheumatoid joint. Immunolocalization of collagenase across the cartilage/pannus junction is described, and in the light of the finding of the specific, small molecular weight beta1-anticollagenase we propose a model of cartilage erosion based on the interaction between collagenase and its natural inhibitors.


Reciprocal impulses in patients with extrasystolia. In 300 patients with extrasystolia and 36 patients with parasystolia a systematic search for reciprocal impulses was conducted with the aid of oesophageal ECG leads. Within 10-min periods, reciprocal impulses were detected in 13 patients (3.9 per cent). Reciprocal impulses occurred in extrasystolias of both functional and organic origin. Exercise contributes to the appearance of postextrasystolic reciprocal impulses. After intravenous injection of 10 mg Obsidan (propranolol hydrochloride) the phenomenon was less marked; in two patients, Obsidan oral medication (40 mg three times daily) led to the disappearance of reciprocal supraventricular paroxysmal tachycardia elicited by ventricular extrasystolia.


Distribution of F-bodies, heterochromatin, and nucleolar organizers in the genome of the central mudminnow, Umbra limi. Due to the very limited amount of knowledge available on the cytochemistry and architecture of fish chromosomes, an extensive banding study was carried out on chromosomes of the central mudminnow, Umbra limi. Through the use of fluorescent staining, C-banding, and silver staining, the chromosomes were characterized, and an idiogram was constructed showing the location of C-band heterochromatin, F-bodies, and the nucleolar-organizer regions. G-banding was attempted but was unsuccessful. Results of fluorescent staining with 33258 Hoechst and quinacrine HC1 raised some interesting questions concerning the cytochemistry of U. limi chromosomes, as well as the specificity of these stains.


Infant day care as a treatment intervention: a follow-up comparison study. Three-year-old children who had been in an infant day care treatment program were compared with matched normal children who were entering regular day care for the first time at age 3, to evaluate the effects of early day care intervention. All children were assessed on general pathology, play, socialization, and separation variables in arrival, play, and mealtime situations and were naturalistically observed in the day centers. No significant differences between groups were found on any of the variables within situations or across situations, supporting the hypothesis that the treatment intervention supported major positive emotional developments and that the early separations were not detrimental in effect. Significant differences between the two groups on clusters of variables suggest patterns in coping and disturbance style specific to the control group and significantly different from the day care group.


Patterns of silver staining of human chromosomes. The chromosomes of twenty individuals with normal karyotypes were studied to determine the patterns of staining with the Ag--AS technique. These patterns were shown to be variable from one individual to another, but characteristic and constant within each individual. In addition, one patient with chronic lymphocytic leukemia was studied, and shown to have an Ag--AS staining pattern that was distinctly different from that of normal subjects.


[Short- and long-term prognosis of cardiac arrest in acute myocardial infarction (author's transl)]. Of 80 patients with acute myocardial infarction who had a cardiac arrest without shock 42 (52.5%) were resuscitated in a cardiological intensive care unit. Twenty-six were finally discharged from hospital. After an average of four years, 21 patients were still alive. Prognosis of primary ventricular fibrillation, the most frequent cause of circulatory arrest (51) was more favourable than that of primary asystole (23 patients). The younger the patient the better the prognosis: the average age of the 21 who survived for several years was 12 years less than those who had died. Follow-up examination indicated that 13 had signs of heart failure. Ventricular extrasystoles were demonstrated by ECG in 11. Eight had depressive episodes. It is likely that a further decrease in death-rate can be achieved only if the interval between onset of infarction and admission to an intensive care unit can be shortened.


[False diagnosis of "osteoporosis" in diffuse multiple myeloma (author's transl)]. Multiple myeloma is very frequently diagnosed too late. In ten patients in whom the diagnosis was ultimately made in the course of one year, the early symptom of back or skeletal pain was at first most frequently misdiagnosed as osteoporosis and so treated. Such symptoms as loss of weight, increased sedimentation rate and increase in gamma-globulins should have led to further tests, for example sternal puncture, radiological survey of the entire skeleton, and immuno-electrophoresis.


Automatic detection and localization of epileptic foci. A new method of automatic EEG analysis (ASD or automatic non-stationarity detection) derived from a parametric EEG model (autoregressive filter model) and based on inverse filtering, has been applied to scalp and subdural EEGs of epileptic patients. By this method it was possible to detect the occurrence of transient non-stationarities such as paroxysmal patterns of activity (spikes, spike-and-waves) characteristic of inter-ictal EEGs. The ASD has been implemented in a general purpose digital computer. The method allows: (a) statistical evaluation of paroxysmal patterns; (b) multi-channel analysis, where the interrelationships of different derivations are quantified and displayed by means of a clinically useful spatial map. The application of the ASD method to scalp and subdural EEGs, simultaneously recorded, has revealed that a number of transient non-stationarities detected in the scalp by the program yet not by visual inspection, coincide with clear paroxysmal patterns in subdural derivations. In this way conventional definitions of characteristic paroxysmal patterns at the scalp are put in question. Identical conclusions regarding the localization of an epileptogenic zone in 4 patients were obtained either using the conventional methods of recording electro-clinical seizures in EEGs of long duration or applying the ASD method to short EEG epochs (100 sec long, at most).


Automated analysis of sleep in the rat.

Automated analysis of sleep in the rat. The four sleep states of the rat were identified by an on-line or a rapid off-line method of automated analysis of the ECoG and EMG.


On the volume conduction in human skeletal muscle: in situ measurements. Volume conduction was studied in situ in human skeletal muscle, using a "single fibre multielectrode". The multielectrode was inserted perpendicular to the fibre axis, and the same single fibre action potential was recorded consecutively from 4 neighbouring leading-off surfaces. The transfer functions of 10 sections of muscle tissue lying between pairs of neighbouring electrodes were calculated from the Fourier transform of the input and output, i.e. the action potential recorded by the two neighbouring leading-off surfaces. The transfer function had one dominant pole, and was therefore modelled by a section of lumped RC elements The average gain factor and average time constant calculated from the transfer function for a muscle tissue section of 76 mu length were 0.55 and 69 musec, respectively. The radial attenuation of the different sinusoidal components of the extracellular action potential was calculated, and was found to be greater than previously reported in the literature, particularly for the slow frequency components. As an explanation of these findings, the possibility of a greater anisotropy of the muscle tissue than previously reported is discussed.


EEG power spectral changes secondary to L-DOPA treatment in parkinsonian patients: a pilot study. Clinical EEG evaluation and power spectral analysis were performed on 12 parkinsonian patients before and during L-DOPA treatment. The EEG evaluation did not disclose any significant effects of L-DOPA treatment. Spectral analysis disclosed normal interlobar and interhemispheric power differences and an increase in delta and slow-theta power in the right temporal as compared to the right occipital lobe before L-DOPA treatment was initiated. L-DOPA treatment significantly increased alpha power in both occipital lobes, decreased theta power in the right temporal lobe, accentuated power differences in the theta and alpha bands between temporal and occipital lobes and attenuated beta power differences in those areas. This work is intended as a pilot study in the quantitative EEG evaluation of Parkinson's disease and L-DOPA treatment.


Increased upper airway resistance to breathing during sleep in the cat. At  the onset of sleep, upper airway resistance shifted to higher levels which were maintained throughout sleep. With each inspiration, there was a decrease in upper airway resistance. These respiratory changes in resistance were smaller in wakefulness (on low baseline resistances) than those in nonrapid eye movement sleep (on high baseline resistances). In rapid eye movement sleep, modulations with inspiration were diminished and were intermittently absent, and baseline resistance was high.


Computer rejection of EEG artifact. II. Contamination by drowsiness. As part of an effort to automatically measure a background EEG baseline against which changes due to therapy or experimental manipulations may be measured, algorithms to detect EEG patterns associated with drowsiness have been developed and objectively evaluated. The decision of drowsiness is tentatively based upon changes in simple signal features, including increased ratios of both delta-band to alpha-band and theta-band to alpha-band spectral intensity as compared to thresholds automatically determined from a waking calibration period. Several heuristic criteria are then required to reach a final decision. Thirty-one normal and abnormal, 3-minute, 8-channel clinical EEG recordings containing drowsiness were scored by 5 expert scorers. Out of a total of 106 events labeled drowsy by at least one judge, 85 were found by a consensus of 3 or more of the 5 experts. On the 20 recordings not used for training the decision thresholds (testing data set), the system found 84% for the 85 episodes found by the consensus, and 89% of the 62 episodes found by all 5 scorers. Only one event was found by the system which was not found by any scorer, or which did not border on a consensus-defined episode of drowsiness. This performance is adequate to justify inclusion of these algorithms into a previously described real time EEG analysis system, ADI-EEG, allowing integration of the decisions of the separate subsystems for detection of artifact, sharp transients and drowsiness.


Evoked potentials and brain maturation in hyperactive and normal children. Auditory evoked cortical potentials in 39 hyperactive children in the age range of 72--144 months and 39 age sex matched normal control children were studied. The importance of careful control of age when studying evoked cortical potentials in hyperactive children is illustrated. Evidence is presented for two independent evoked potential components (P1--N1 and P2--N2) both of which show abnormal changes with maturation in hyperactive children. It is suggested that changes in these two evoked potential components may reflect abnormal development of two quasi-independent neural substrates in the hyperactive child. The 77% successful classification by means of discriminant function analyses (utilizing two evoked potential components) suggests potential usefulness of this objective laboratory measure as an aid to the diagnostic classification of hyperactive children.


Volume conduction and EEG measurements within the brain: a quantitative approach to the influence of electrical spread on the linear relationship of activity measured at different locations. When recording referentially brain field potentials with several electrodes at relatively small tip separations, a linear relationship between the simultaneously recorded signals may arise solely as a result of volume conduction (electrical spread). A method is described to quantify the linear relationship due to electrical spread in a situation with independent neuronal sources. In rat under urethane anaesthesia, records were made during theta activity in the hippocampus with two electrodes against a reference with electrode tip separations between 0--3 mm. Frequency analysis of EEG epochs and computation of coherence were carried out. As an estimate of linear relationship between the recorded signals due to electrical spread the mean value of coherence (cohm) of a frequency band outside the range containing most power of theta rhythm was calculated. The results show a fairly constant decay of cohm at increasing electrode separation, reaching a value of 0.1 at a distance varying between 0.8--1.4 mm. This means that neurones at a distance of 0.4--0.7 mm from a recording electrode make a contribution of -25 dB to a recorded signal of 0 dB. The results of a simple model of volume conduction producing linear relationship between two recorded signals are in good agreement with the experimental results. The influence of linear relationship of the activity of neurones on volume conduction properties and on coherence is discussed.


Relationship between amplitude of pattern displacement and visual evoked potentials. Thirteen subjects looked at a 7 mm2 black and white checkerboard which was displaced through 0.25, 0.50, 0.75 and full pattern displacement. One hundred and twenty eight averages were taken for each displacement and the peak to peak amplitude of the visual evoked potential was measured. This showed a linear relationship between pattern displacement and size of the evoked potential. Latency was unaffected for 0.25, 0.50 and 0.75 displacement but was significantly longer for the full pattern stimulation.


The effects of phencyclidine on visually evoked potentials of rhesus monkeys. Phenyclidine (Sernylan) was given intramuscularly to 12 rhesus monkeys and electroencephalograms and visually evoked potentials to photic stimulation were recorded. Phencyclidine has profound effects upon VEP's, particularly a marked enhancement of positive components maximal around 100 msec after stimulus presentation. Most likely these effects are related to the level of surgical anesthesia, Stage III, phase I. A correlation was suggested between the increased amplitude of VEP's and the psychotic clinical manifestations of visual perceptual distortions. Physicians and investigators should be aware of the striking effects of this compound, now widely used as a street drug "angel's mist" of "angel's dust", on neurophysiological functions.


Hemispheric differences in the auditory evoked response. The separation of the 80-250 msec portion of the AER to click stimulation into N1P2, produced by large areas of cortex, and the T complex, probably produced by secondary auditory cortex, was recently described (Wolpaw and Penry 1975). The present study investigated the ipsilateral vs. contralateral and right vs. left hemispheric differences in N1P2 and T complex latencies and amplitudes. One msec clicks at 60 dB above threshold were presented at 4.7 sec intervals monaurally to 32 normal adults and binaurally to 13 of the 32. AERs were recorded from vertex and temporal referred to a balanced non-cephalic reference electrode. For monaural stimulation, N1P2 and T complex amplitudes were significantly greater and T complex latency was significantly less over the contralateral and right hemispheres. The ipsilateral vs. contralateral and right vs. left hemispheric amplitude differences were significanlty greater for the T complex than for N1P2. Binaural results, which provided in additional measure of right vs. left hemispheric differences, were in agreement with the monaural findings. The results are consistent with neurophysiologic and behavioral findings in regard to ipsilateral vs. contralateral and right vs. left hemispheric differences and support the hypothesis that the T complex is produced by secondary auditory cortex.


Estimation by radioimmunoassay of VH determinants (Aa1) associated with rabbit T lymphocytes. A radioimmunoassay with specificity for the a1 allotype determinant of rabbit immunoglobulin (Ig) heavy chains was used for the quantitation of VH in detergent extracts of lymph node cells from a1a1/b4b4 rabbits. B cell-derived Ig in the same extracts was estimated with a radioimmunoassay specific for L-chain. About 2 X 10(5) molecules/cell was found by both assays when assuming a molecular weight of 1.5 X 10(5) dalton. Extracts prepared from lymphocytes depleted of B cells (surface Ig+ cells) were also found to contain corresponding amounts of a1 and L-chain, with values varying from about 10(3) to 10(4) molecules/cellmthe estimates were not influenced by the presence of inhibitors of proteolytic enzymes. Supernatants, obtained after keeping the lymphocytes in culture for 16 h, were analyzed by the same methods. Again, matching amounts of a1 and L-chain were found in supernatants from nonfractionated as well as from B cell-depleted populations. The results obtained indicate that T cells do not carry or produce an excess of a1 compared to L-chain, as would have been expected if the T cell antigen receptor carried the same variable region as the Ig heavy chain while being otherwise composed of nonimmunoglobulin-like structures.


Mechanism of target cell lysis of cytolytic T lymphocytes. I. Characterization of specific lymphocyte-target cell conjugates separated by velocity sedimentation. Differential velocity sedimentation was applied for separating alloimmune T lymphocytes bound to target cells (TC) from free lymphocytes. Maximal size differences between lymphocytes and TC were achieved a) by isolating the fraction of small peritoneal lymphocytes (SPL) from an alloimmune peritoneal cell population, and b) by selecting large tumor cells as TC. Under the conditions used, most of the conjugates formed at room temperature consisted of one SPL bound to one TC, and adequate separation of bound from free SPL could be achieved within less than 5 min. Functional studies of the conjugate-enriched fractions showed that a minimum of 60% of TC-bound SPL were indeed cytolytic. Conjugate-depleted fractions, however, were still lytic, suggesting that not all effector cells formed stable conjugates at room temperature. Transmission and scanning electron microscopy studies revealed that binding between SPL and TC was achieved through interpenetrating membrane projections and was characterized by point and broad zone contacts. When lysis was allowed to proceed, prominent changes of the TC membrane morphology, including loss of microvillous projections, appearance of localized blebs, pseudopod-like projections, and membrane defects were documented.


VH and L-chain allotype determinants of rabbit IgG and IgA estimated by a semi-automatic, modified Farr-type radioimmunoassay;. A sensitive radioimmunoassay for rabbit immunoglobulin allotypes has been developed. Separation of antibody-bound and free antigen was accomplished by salt precipitation with ammonium sulfate, followed by collection and wash on glass fiber filters by means of a semi-automatic cell harvester. The efficiency of the collection was increased by the addition of nonionic detergent which was salted out together with the precipitated reagents. The presentation of the a 1 allotype, located in the VH region, was found to be different on IgG and IgA molecules.


Induction and characterization of "autologous" anti-isiotypic antibodies. Upon immunization with phosphorylcholine (PC), conventional BALB/c mice produce anti-PC antibodies bearing predominantly the idiotype characteristic of the BALB/c PC-binding myeloma protein TEPC 15 (T15). To investigate whether BALB/c mice are able to produce antibodies to the autologous T15 idiotype, conventional (T15-positive) and neonatally suppressed (T15-negative) BALB/c mice were immunized with purcific for the T15 idiotype. However, anti-T15 antibodies were more readily induced in neonatally suppressed mice which in turn produced higher anti-T15 titers than conventional mice. Such "autologous" anti-T15 antibodies are able to (a) change the idiotypic pattern of the anti-PC response of conventional BALB/c mice in situ and, (b) inhibit the induction of an anti-PC response in vitro by spleen cells from T15-positive but not T15-negative BALB/mice. Thus, BALB/c mice are capable of producing anti-T15 antibodies upon immunization with an isologous myeloma protein which bears the autologous T15 isiotype. We suggest that isiotypes should not be strictly considered as "self-antigens" since the dramatic increase in their concentration, subsequent to antigen stimulation, might confer upon them immunogenic properties not shared by self-antigens.


Antigen recognition by T cells activated in the mixed lymphocyte reaction: specific binding of allogeneic cell material after removal of surface-bound antigen by trypsin. T cell blasts activated in the mixed lymphocyte reaction (MLR) carry on their surface stimulator alloantigens which can be removed by treatment of the blasts with trypsin. After overnight incubation in trypsin-free medium, the treated cells exhibit specific alloantigen binding ability: they bind much more effectively cellular material, either obtained from the corresponding MLR supernatant or released by nitrogen cavitation from fresh cells from the stimulating strain, than that from an unrelated H-2 different strain. Trypsin-treated cells which have been incubated in the presence of low concentrations of puromycin are unable to bind stimulator cell fragments.


Suppressor cell induction in vitro. III. Antigen-specific suppression by supernatants of suppressor cells. Antigen-specific suppressor T cells induced in vitro release, after a further period of culture in vitro with antigen factors into the supernatant which have suppressor activity. These suppressor factors (SF) have the same antigen specificity as the suppressor cells (SC). SF only works on the early phase of thymus-dependent responses in cultures. SF inhibits thymusdependent IgM, but not thymus-independent IgM responses in vitro. SF is is destroyed by proteolytic enzymes, and inactivated at 80 degrees C for 30 min. The release of SF from SC is dependent on the presence of antigen and metabolically active cells.


Human chorionic gonadotropin, its receptor and mechanism of action. Human chorionic gonadotropin (HCG) is a glycoprotein hormone consisting of two noncovalently bonded subunits, alpha and beta. The hormone can be dissociated and reassociated. Whereas the individual subunits do not show any receptor binding activity, the reconstituted molecule is almost fully active. The amino acid and carbohydrate sequences in hCG-alpha and hCG-beta are described. There are in all seven carbohydrate units, four complex asparagine-linked and three serine-linked short oligosaccharide chains. The sequential removal of monosaccharides from the carbohydrate moiety of the hormone results in derivatives that bind to the cell surface receptors but inhibit the hCG-induced accumulation of cAMP. The derivatives, however, still are able to produce steroidogenesis maximally. The data raise the possibility of other mediator(s) of the hormone action in addition to cAMP. The hCG/LH (luteinizing hormone) receptor has been labeled by the incorporation of N-acetyl-D-1-[14C] glucosamine and also by the selective incorporation of 125I or 131I. Using 131I-labeled bovine corpus luteal plasma membranes, a method for the purification of the receptor to homogeneity has been developed. The purified receptor has properties similar to the membrane-bound receptor. Availability of the purified receptor offers newer approaches to the study of molecular mechanisms of polypeptide hormone action.


[Exocrine function of the pancreas under the effect of different doses of hexachlorocyclohexane]. Experiments conducted on albino rats poisoned with different doses of hexachlorocyclohexane (HCCH) demonstrated that large doses of this compound (1/3 DL50) following their single introduction were capable of inhibiting the secretion of the amylase, lipase, and proteases of the pancreas. Of a prolonged action of relatively small doses of HCCH (1/20 and 1/50 DL50) is characteristic an initial inhibition and subsequent stimulation of the exosecretory function of the pancreas. A definite inverse relation between the amount of the introduced HCCH dose and the duration of the pancreatic inhibition period was noted.


[Paralysis of the intrinsic muscles of the hand]. Palliative treatment of intrinsic muscle paralysis aims at correcting the claw deformity and improving prehension. This treatment will vary according to whether the claw can be actively corrected or not, if M. P. hyperextension is blocked. In the first category it suffices to maintain M. P. joint flexion either by capsulodesis or tenodesis so that the long extensors can extend the interphalangeal joints. Tendon transfers are useful only to reinforce the flexion force of the fingers if there is an associated long flexor tendon paralysis. These transfers should be fixed to the proximal part of the proximal phalanx. If the claw is not actively correctable, associated cutaneous, tendinous or joint lesions must co-exist and these will demand priority treatment. Tendon transfers, if possible, aim to extend the distal phalanges and should be fixed distally on the extensor expansion, knowing however that this carries the risk of swan neck deformity.


[Histoautoradiographic examinations of benign and malignant cells and tissues during therapeutic interventions in gynecology]. The alteration of the proliferation activity of different tissues of the female genital tract was studied with the histo-autoradiographic method. Tissue samples from 1. the normal endometrium before and under treatment with the sequential oral contraceptive Ovanon, 2. carcinomas of the vulva before and under treatment with Bleomycin, 3. cervical carcinomas stage I-III before and under Co 60-radiation, and fluid material of cystic ovarian tumors and effusions before and under treatment with alkylating agents were incubated in 3H-thymidine. Morphologic and histo-autoradiographic alterations were observed during all investigations. It was demonstrated that the histo-autoradiographic method 1. gives informations about the proliferation activity of tissues of the female genital tract, 2. quantifies the success of treatment, 3. and allows prognosis about therapeutic results, as it was shown in the case of radiation sensitivity of cervix carcinomas.


Immunologic tolerance and immunopathology. Immune tolerance has an antigen-directed specificity analogous to that of immune responsiveness. Studies have shown the direction of response-immunization or tolerization-is often dependent on the physical form and the dose of antigen, and that the two major lymphocyte classes, B cells and T cells, have different tolerance requirements. The critical relationships of these findings to autoimmunity are explored.


Carcinogenic action of polycyclic hydrocarbons in animals and in man. Basic data on the carcinogenic action of polycyclic aromatic hydrocarbons (PAH) in animals and in man are presented, mainly from the author's own experience. Benzo[a]pyrene (BaP), a powerful carcinogenic agent widely spread in the human environment, can be considered an indicator of PAH. Quantitative spectral-fluorescent methods developed by the author and his co-workers were used to study the circulation of BaP in the atmosphere, hydrosphere, soil, plants, animals and humans. Accumulation and degradation of PAH, as well as the background level of BaP, in the environment are described. The results were used as a basis for establishing health standards and other preventive measures against cancer.


Analysis of automobile exhaust condensates. 1. On the basis of figures for the production of PAH during the Europa drive cycle by 100 passenger cars and those for the consumption of petrol in the Federal Republic of Germany, an annual emission of 1,850 kg benzo[a]pyrene from petrol engine vehicles has been calculated. 2. The carcinogenic effect of benzo[a]pyrene accounts for only 9% of the total activity of exhaust condensates. 3. The amounts of other known carcinogenic PAH, such as benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[j]fluoranthene, indeno[1,2,3-cd]pyrene and dibenzo[a,h]anthracene are shown in Table 2. (table: see text). Assuming that there is no significant promoting or hyper-additive effect, it can be estimated that these six known carcinogenic PAH contribute about 10-15% of the total carcinogenicity. 4. Six unknown PAH were found: cyclopenteno[cd]pyrene (mol wt 226), methylenebenzo[a]pyrene (mol 264), methylenebenzo[e]pyrene (mol wt 264), methylenebenzo[ghi]perylene (mol wt 288), PAH mol wt 300A and PAH mol wt 300B. It is reasonable to assume that these unknown PAH account for the predominant part of the carcinogenic effect. Biological tests with these pure substances are being undertaken by Drs Pott, Pfeiffer and Habs. 5. It has been shown that almost all of the carcinogenic effect of automobile exhaust condensates is due to PAH. To support this claim, the carcinogenic effects of the exhaust condensate should be compared with those of a mixture of the known and unknwon PAH in the same proportions as are found in the exhaust condensate. The gas chromatogram of such a mixture is shown in Figure 6.


Experimental results with percutaneous applications of automobile exhaust condensates in mice. The induction of squamous-cell carcinoma and papillomas in mice after 80 weeks' skin painting with different doses of automobile exhaust condensates and their fractions is described. The results demonstrate that the carcinogenic activity of the condensate resides in its polycyclic aromatic hydrocarbon fractions. Benzo[a]pyrene accounts for approximately 9% of the activity of the condensate. The sebaceous gland suppression test, used as a screening method, is predictive of carcinogenicity and is positive for those condensate subfractions which contain carcinogenic PAH.


Syncarcinogenic action of polycyclic hydrocarbons in automobile exhaust gas condensates. The carcinogenic action of the hydrocarbon mixture predominantly found in condensates of automobile exhaust gas can be attributed almost exclusively, on the basis of the results presented here, to a group of substances (PAH) with an insignificant weight proportion. It was not possible to demonstrate an inhibitory action of most of the weak-to-inactive hydrocarbons; on the contrary, an additive effect of the two types could be observed. At very high doses (almost 10 times higher than the highest doses selected in the rest of the trial) the group of substances which were supposed to be non-carcinogenic also proved to be biologically effective. Subsequent individual studies on the same animal strain should clarify whether this action is to be attributed to one or two representatives of this group, e.g., benzo[e]pyrene or chrysene.


Oncogenic interaction of carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons in mice. To evaluate possible interactions between PAH occurring in automobile exhaust condensates with regard to their tumour forming potency, the following experiments were performed. Six different doses of benzo[a]pyrene (3-100 microgram) and of dibenzo]a,h]anthracene (2-75 microgram) and mixtures thereof were tested subcutaneously on female NMRI mice. In addition, mixtures of 10 non-carcinogenic hydrocarbons were applied: benzo[e]pyrene, benzo[a]anthracene, phenanthrene, anthracene, pyrene, fluoranthene, chrysene, perylene, benzo[ghi]perylene and coronene. Mixtures of all 12 PAH were also applied. The proportion of PAH in all mixtures used was the same as in automobile exhaust condensates; benzo[a]pyrene was used as reference substance. The most important results were as follows: 1. Small doses of dibenzo[a,h]anthracene have a greater tumour promoting effect than do comparable doses of benzo[a]pyrene. Increased doses increase the effect of benzo[a]pyrene more than that of dibenzo[a,h]anthracene. 2. The mixture of benzo[a]pyrene and dibenzo[a,h]anthracene is 1.4 time more active than dibenzo[a,h]anthracene alone. 3. The mixture of all PAH has a lower efficacy than dibenzo[a,h]anthracene alone, amounting to only 0.03 that of dibenzanthracene; however, the activity of dibenzo[a,h]anthracene within the mixture of the 12 PAH increases by a factor of 3.1. 4. The activity of a mixture of dibenzo[a,h]anthracene and benzo[a]pyrene depends to about 40% on dibenzo[a,h]anthracene; and that of 12 PAH to 30% on dibenzo[a,h]anthracene alone or to 80% on a mixture of dibenzo[a,h]anthracene and benzo[a]pyrene.


Tumours in mice after subcutaneous injection of automobile exhaust condensates. Automobile exhaust condensate (AEC), either mixed with benzo[a]pyrene (BaP) or suspended or dissolved in tricaprylin, was injected subcutaneously into NMRI mice in a series of experiments. The addition of AEC decreased the incidence of tumours which developed with 30, 90 and 270 microgram BaP. Reduction of tumour incidence was proportional to the amount of AEC added. With an injection of 10 microgram BaP, the latent period was greatly increased when AEC was added, but the occurrence of tumours was the same. Components of AEC appear to inactivate BaP, at least temporarily. In further experiments AEC and nine fractions thereof were injected subcutaneously into mice. The fraction comprising only polycyclic aromatic hydrocarbons (PAH) induced the highest incidence of tumours. In contrast, when it was administered in combination with other fractions the PAH fraction was less active. Application of the products of further fractionation of PAH showed that polycyclic compounds with seven or more rings can also induce tumours in this model.


The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). The history and characteristics of two cells lines developed from primary explants of pupal tissue from the insect, Spodoptera frugiperda (J.E. Smith), are described. One cell line, IPLB-SF 21, was developed with hemolymph-supplemented medium and has been maintained continuously on the medium. The second cell line, IPLB-SF-1254, was developed with a medium containing a combination of vertebrate sera plus hemolymph and was adapted to hemolyphn-free medium at the 6th passage. The IPLB-SF-21 cell line has a population doubling time of 26 to 30 hr; the doubling time of the IPLB-SF-1254 line is 36 hr. The chromosomal morphology and distribution was typical of other lepidopteran cell lines. Serological studies showed that both cell lines have at least one antigen which also is common is tissue antigens from pupae of Spodoptera frugiperda.


Ovine cells: their long-term cultivation and susceptibility to visna virus. Sheep choroid plexus (SCP) cells have been subcultured more than 120 times and have undergone over 300 cell generations. These fibroblastic-appearing SCP II-B cells contain ovine-specific antigens, have an absolute plating efficiency of 23 to 28% and are as susceptible to visna virus infection and virus-induced cytopathology as their low passage level counterparts. Cultures of low, relatively high and high passage level SCP cells produced equivalent amounts of visna virus at similar rates when infected with equal amounts of visna virus. The passage level of the SCP II-B cells, their elapsed number of cell generations, their possession of ovine-specific antigens and their full susceptibility to visna virus allow these cells to be considered an established line of sheep cells.


Variable region (Fv) determinants on mouse lymphocytes. Antisera have been raised to the Fv fragment derived from a mouse alpha/lambda myeloma. These had different reactivities for Fv and mouse immunoglobulin and two of them appeared to detect v region framework determinants on normal mouse immunoglobulins. A large proportion of mouse lymphocytes were shown to have Fv determinants on the surface which was part of the B-lymphocyte antigen receptor. Most of the reacting cells were B lymphocytes. Fv determinants were not detected on the majority of T lymphocytes including cells with known T-helper cell activity. It is concluded that in mice, T cells do not express immunoglobulin v region framework determinants on their surface.


The effects of chemical modification on the antigenicity of human and rabbit immunoglobulin G. In order to characterize the precise structure within human and rabbit IgG molecules against which 'general' rheumatoid factors are directed, an immunochemical comparison has been made of the effects of the selective substitution of specific amino acid side-chains on various types of antigenicity exhibited by human and rabbit IgG. The epsilon-amino groups of lysine residues have been substituted by citraconylation and carbamylation; whilst tyrosine residues have been substituted by nitration with tetranitromethane. In this manner, evidence has been obtained which indicates that the autoantigenic determinants of human IgG are structurally distinct from species-specific ones and from certain Fc-located allotypic markers (Gm(a) and Gm(x)). It is also concluded that lysine residues are probably not involved in the site of IgG reactivity with 'general' rheumatoid factors, in contrast to tyrosine residues which appear to be implicated in the activity of human but not rabbit IgG.


Production and specificity of antibodies against the aminoterminal region in type III collagen. A cross-linked fragment (peptide T1X) with a molecular weight of 13,000 could be isolated from a tryptic digest of insoluble type III collagen of calf skin. Peptide T1X was conjugated on to bovine serum albumin by glutaraldehyde and used for immunization of rabbits. The antisera reacted in passive haemagglutination and radioimmune assay with peptide T1X, type III collagen and its constituent alpha1(III) chain. Little or no reaction was observed with type I collagen and alpha1(I) chain. While rabbit antisera to neutral salt-soluble type III Collagen also showed a strong binding for 125I-labelled peptide T1X much less reaction was observed with antisera to type I collagen. The antigenicity of type III collagen was largely destroyed by pepsin treatment suggesting that it resided in non-helical segments. A fragment of peptide T1X produced by digestion with collagenase retained antigenic activity. The data indicated that the aminoterminal region of type III collagen contains strong antigenic determinants located in a non-helical sequence of about sixteen amino acids. Antibodies to these antigenic determinants were purified and rendered specific for type III collagen by immunoadsorption. The antibodies stained in indirect immunofluorescence tests particularly those regions in various connective tissues which are rich in reticulin fibres. Different staining patterns were observed with antibodies to type I collagen.


Non-precipitating guinea-pig antibodies as products of limited recognition of antigenic sites on the ovalbumin molecule. The non-precipitating guinea-pig IgG1 and IgG2 antibodies, produced by administration of an excessive dose of hen ovalbumin (OA), were capable of forming only certain soluble complexes having molar ratios of antibody to antigen lower than 1-5:1, which were lower than the minimum ratio of 2-5:1 shown by anti-OA antibodies precipitable with OA. This unusual property of the non-precipitating antibodies was caused by a limited number of antibody molecules capable of binding to one OA molecule simultaneously, since the maximum number of their Fab' fragments binding to one OA molecule was only three and markedly less than the number (7-8) of Fab' fragments of the precipitating IgG1 and IgG2 anti-OA antibodies binding to one OA molecule. These results demonstrate that the non-precipitating IgG1 and IgG2 anti-OA antibodies are those reacting with some particular antigenic sites on OA, a number of the antigenic sites too few to permit insoluble lattice formation.


Studies on the quantitative relationship between specific antigen recognition and manifestation in delayed hypersensitivity. Delayed skin reactions or peritoneal cell migration inhibition were elicited in guinea-pigs or rats with egg albumin, diphtheria toxoid, or tuberculin-purified protein derivative either separately or with various combinations of two antigens. When the skin reactions or migration inhibition with the component antigens alone were relatively strong, the corresponding combination elicited weaker reactions than calculated assuming that the antigens would add upon each other independently, i.e. the non-specific phase was limiting the manifestation. When the reactivity to both components was undetectable or very weak, the combination elicited stronger reactions than calculated, implying synergism of antigen recognition. The combinations of two weakly positive or intermediate reactivities were close to the calculated values in both the diameter of erythema, skin thickness and migration percentage. These results may help in assessing the biological significance of findings in delayed hypersensitivity: even minor differences in strong manifestations may mean great differences in terms of specific lymphocytes, while marginal changes of antigen recognition may lead to apparently great changes of manifestation, when very weak reactivities are measured.


The effect of anti-eosinophil serum on skin histamine replenishment following passive cutaneous anaphylaxis in the guinea-pig. Depletion of eosinophils by an antieosinophil serum resulted in the inhibition of histamine reaccumulation into guinea-pig skin sites following IgG1-mediated passive cutaneous anaphylaxis. This suggests that eosinophils may have a regulatory role in the repair mechanism which follows immediate-type hypersensitivity reactions.


The immune response to oxidized ferredoxin. I. Specificity of the response to the amino terminal determinant. Several synthetic peptide analogues of the amino terminal antigenic determinant (ala-tyr-lysile-ala-asp-ser) of oxidized ferredoxin (O-Fd) were tested for their ability to inhibit the complement fixation reaction between O-Fd and homologous rabbit antiserum, and to inhibit the migration of spleen cells from guinea-pigs immunized to O-Fd or to a conjugate of the amino terminal heptapeptide (N7) and bovine serum albumin (N7-BSA). The results of the migration inhibition assay suggest that the tetrapeptide and longer peptides of the native sequence were all recognized and stimulated the production of migration inhibition factor. Peptides modified at the aspartic residue were partially active while the serine modified peptide was not. Modification at the amino end of the heptapeptide had no effect on migration inhibition. As specificity controls, it was shown that the N7-BSA conjugate inhibited migration in O-Fd immunized animals, while O-Fd inhibited migration in N7-BSA immunized animals. The hexa-, hepta-, aspartic-deleted and serine-modified peptides were able to inhibit the complement fixation reaction with O-Fd and specific rabbit antiserum. Inhibition found with the serine-modified peptide and the lack of inhibition with the amino-modified peptide or the di-, tri-, tetra- and pentapeptides indicates the determinant recognized by the rabbit antibodies is either larger or is located nearer the middle of the heptapeptide than the determinant which induced the production of MIF.


Detection and molecular characterization of the thymus-brain antigen in human brain. Anti-human brain sera were found to contain antibodies reacting with determinants present on murine thymocyte plasma membranes. This determinant is borne by the thymocyte-brain antigen of mouse thymocytes. The non species-specific determinant of the thymocyte-brain system is detectable on the thymocyte-brain antigen of human and mouse brain in comparable amounts. In contrast to these findings, the allogenic Thy-1 and the species-specific determinant of this system were only found on the thymocyte-brain antigen of mouse brain but not on the corresponding antigen of human brain. The molecular weight, Stokes radius and diffusion coefficient of the thymocyte-brain antigen of human and mouse brain are in good accordance with the data estimated for the corresponding antigen of murine thymocytes.


Concanavalin A induced histamine release from human basophils in vitro. The site of interaction for concanavalin A (Con-A)-induced histamine release from human basophils was studied in vitro. Blocking the epsilon one determinant (D leads to 1) of IgE with high concentrations of monomer (Fab) anti-Depsilon1 does not significantly inhibit the quantity of histamine released by suboptimum concentrations of Fc specific anti-IgE. This indicates that the monomer anti-Depsilon1 does not have the capacity to sterically hinder the bridging of all of the determinants in the Cepsilon3 and Cepsilon4 domains (Fc'-epsilon-region) of IgE. The monomer anti-Depsilon1 does effectively inhibit release induced by suboptimum concentrations of Con-A. The data indicate that for suboptimum concentrations, Con-A activation is IgE mediated and takes place in the proximity of Depsilon1 and not at the membrane receptor for IgE.


Immune response against two epitopes on the same thymus-independent polysaccharide carrier. 1. Role of epitope density in carrier-dependent immunity and tolerance. Immunogenicity and tolerogenicity of two epitopes (alpha 1-6 and FITC) on the same dextran B 512 carrier were investigated. The following conclusions were made: (1) Both epitopes were thymus-independent and immunogenic and tolerogenic as well. (2) The marked dose differences between the two epitopes with regard to tolerance induction were found to be a consequence of the affinity and the heterogeneity of the responding B cells as well as the epitope density employed for detecting the PFC in a predictable way. (3) The alpha 1-6 response, in contrast to the anti-FITC response, was homogeneous and of low affinity and the number of precursor B cells was low. (4) Different mouse strains were found to be high-, low- or non-responders to alpha 1-6, but all the strains tested responded to the FITC epitope coupled to dextran. (5) Dextran and FITC-dextran were polyclonal B cell activators in the strains tested, irrespective of their ability to respond to the alpha 1-6 epitope. The findings indicate that epitope density and mol. wt of the immunogen as well as Ig receptor affinity for the epitope on the B cells are variables which markedly influence the binding of the immunogen to the specific B cells and therefore affect the delivery of the non-specific triggering signal.


Differences in the maturation of the immune response of A- and A+ rabbits. Good and poor responders respectively for the A antigen. The amount, avidity and specificity of anti-A antibodies produced by rabbits having the A substance in their secretions (A+) and rabbits lacking this antigen (A-), were studied with a series of antigens chemically related to the A substance. Both A- and A+ rabbits produced specific anti-A antibodies but the amount, maturation of avidity and specificity of these antibodies was completely different. These results suggest that the difference between the immune response of A+ and A- animals is not based on a genetically determined inability of A+ rabbits to recognize the self antigen, but rather on differences in the regulatory mechanism(s) controlling the maturation process of the anti-A response.


Differentiation of several isolates of Peptococcus magnus by counterimmunoelectrophoresis. Uniform sonicated suspensions of three clinical isolates of Peptococcus magnus that were morphologically, chromatographically, and biochemically identical were used to produce hyperimmune antisera in rabbits. Similar hyperimmune serum was prepared to a single strain of Peptostreptococcus anaerobius. When these antisera and antigens were tested by counterimmunoelectrophoresis, the three isolates of Peptococcus magnus were immunologically distinct. None of the antisera to Peptococcus magnus formed precipitin lines with Peptostreptococcus anaerobius.


Transfer of delayed hypersensitivity in mice to microbial antigens with dialyzable transfer factor. Dialyzable Lawrence-type transfer factor was prepared from the spleen cells of CF1 mice inoculated with Coccidioides immitis- and Candida albicans-killed vaccines and with live Mycobacterium tuberculosis vaccine (BCG). These preparations were shown to transfer antigen-specific cell-mediated immunity to naive mice, as measured by the delayed skin test and footpad-swelling methods. Reactivity could be demonstrated when the test antigens were given 24 h after the transfer factor, but not when they were given simultaneously. Coccidioides-specific transfer factor was shown to be sensitive to Pronase and resistant to trypsin and ribonuclease. A preparation of BCG transfer factor was sensitive to snake venom phosphodiesterase.


Incidence of exotoxin production by Pseudomonas species. Pseudomonas aeruginosa exotoxin A has been shown to catalyze the transfer of the adenosine 5'-diphosphate (ADP)-ribose moiety of nicotinamide adenine dinucleotide onto elongation factor 2, resulting in the inhibition of mammalian protein synthesis. The enzymatic activity (ADP-ribosyl [ADPR]-transferase) is thought to account for the toxicity of exotoxin A. The distribution of the expression of exotoxin A within Pseudomonas species was examined. Laboratory strains as well as clinical isolates of Pseudomonas aeruginosa were tested. The production of exotoxin A was determined by assaying for ADPR-transferase activity in dialyzed frozen (-20 degrees C) and thawed cell-free supernatants from 22-h cultures or in 10-fold-concentrated supernatants. In addition, toxin production was detected immunologically using a modified Elek test. Exotoxin A production was detected in approximately 90% of the 111 isolates of P. aeruginosa. In contrast, none of the other species of Pseudomonas examined produced exotoxin A detectable by either ADPR-transferase activity or immunological reactivity.


Antigenic relationships among thiol-activated cytolysins. Cereolysin, streptolysin O, and perfringolysin O formed precipitin lines that completely fused when reacted with horse antitetanolysin by Ouchterlony immunodiffusion and formed precipitin lines that showed either partial or complete fusion when diffused against horse antistreptolysin O or antiperfringolysin  O.


Resistance to tolerance induction and age-dependent cellular changes in SJL mice. SJL mice became resistant to tolerance induction between the 2nd and 6th week of life. The effect on tolerance induction of radioresistant accessory (A) cells does not depend on the age of the donor, i.e. is the same whether the A-cell donor is 3 or 12 weeks old. In the thymectomized recipient, reconstituted with spleen cells of 12-week-old donors, resistance to tolerance is greater than in the corresponding intact animal. Resistance to tolerance is due to a change in the T cells, occurs at a different rate in the T cells of thymus and spleen, and my be due to change in the relative proportion of different T-cell subpopulations.


Further characterization of alkaline phosphatases of human kidney and urine. Human kidney alkaline phosphatase (AP) react with antisera to liver and intestinal AP. The respective kidney enzymes are designated as KLi and KI. Comparison with liver AP showed that KLi was salted out at the same ammonium sulfate concentration (66%) as the liver enzyme; KLi was antigenically identical with liver AP; the electrophoretic mobility of KLi was that of a gamma-globulin whereas liver AP was an alpha2-globulin. the liver enzyme was lighter (130,000 daltons) than KLi (150,000 daltons). KI and intestinal AP shared most properties: antigenically they were indistinguishable; both contained two components one of which was 80,000 daltons, the other was heavier (120,000 daltons for KI, 130,000 daltons for intestinal AP). Urinary AP consisted only of the lighter component (80,000 dalton). Methods for partial separation of the three kidney APs are given.


Effect of lung tissue on the inhibitory actions of isoprenaline, theophylline, disodium cromoglycate and PGE1 on antigen-induced mast cell degranulation. Isoprenaline, disodium cromoglycate and PGE1 poorly inhibit antigen-induced degranulation of sensitised rat peritoneal mast cells, but in the presence of chopped rat lung their inhibitory activities were markedly enhanced. The chopped lung preparation had no effect on degranulation in the absence of an inhibitor and did not enhance the weak inhibitory potency of theophylline. Lung tissue suspensions pretreated with isoprenaline released a factor into the supernatant which enhanced the action of isoprenaline on peritoneal mast cells. The activity of this supernatant factor was destroyed by boiling for 5 min. Supernatant fluid from untreated lung tissue did not itself reduced antigen-induced degranulation or enhance the inhibitory potency of isoprenaline.


Alpha-1 antitrypsin (AAT) and alphafoetoprotein (AFP) in sera of patients with germ-cell neoplasms: value as tumour markers in patients with endodermal sinus tumour (yolk sac tumour). Serum alphafoetoprotein (AFP) and serum alpha-1 antitrypsin (AAT) were determined in 24 patients with germ-cell neoplasms of the gonads and extragonadal sites and in two patients with hepatocellular carcinoma. In the majority of the patients serial determinations were performed. All seven patients with testicular seminoma and four patients without evidence of active disease had normal levels of serum AAT and AFP. The remaining 13 patients with germ-cell neoplasms had tumours containing endodermal sinus tumour (yolk-sac tumour) elemetns. All these 13 patients had elevated levels of serum AFP and the levels were high or very high in most cases. Nine of these 13 patients had raised serum AAT, although the elevation above normal levels was only slight in a number of cases. When serial determinations were performed serum AAT levels frequently followed the pattern of serum AFP levels, but the AAT levels were frequently within normal limits and therefore the interpretation of the results was difficult, and much less reliable as compared with those for serum AFP. The elevation of serum AAT levels following the recurrence of the tumour was found to occur much later and was much less marked than elevation of serum AFP, which occurred early, showed a large rise and was a reliable marker of tumour recurrence in patients with germ-cell neoplasms containing endodermal sinus tumour elements. It is therefore considered that, although there is good evidence that serum AAT is produced by endodermal sinus tumour elements, serum AAT is not a useful monitor of disease activity in these patients, especially when compared with serum AFP, the value of which is well recognized. Serum AAT may be a useful tumour marker in patients with hepatocellular carcinoma, and this aspect should be investigated further.


Immunologic aspects of leprosy with special reference to the circulating antispermatozoal antibodies. Macroscopic sperm agglutination in gelatin, sperm immobilization and tanned red cell hemagglutination tests could detect antispermatozoal antibodies respectively in 41%, 37%, and 23% sera of 35 leprosy patients, including 5 female cases. Interestingly, all of the above tests were positive in one serum from a female patient with borderline leprosy. Sperm antibodies were detected in both lepromatous and tuberculoid forms of leprosy by the above three technics and no significant difference was observed in their incidences among the two groups of patients. A three dimensional correlation was observed in 57% of 42 tests performed with 14 sera. Head-to head type of agglutination was the predominant feature of spermagglutination observed in the sera of these patients. In the control group, only 1 of 50 normal fertile male showed a positive spermagglutination test. Not one in this group showed positive sperm immobilization and tanned red cell hemagglutination tests. Antihuman globulin consumption test, presumably a very sensitive test, was also employed to demonstrate sperm-specific antibodies in the sera of these leprosy patients. These antibodies were adsorbed on the surface of the normal donor's spermatozoa when the latter were incubated with the patients sera. Antispermatozoal antibody could be demonstrated by this sensitive technic in the sera of two female patients. Moreover, antihuman globulin was consumed more intensely by the antispermatozoal antibodies present in the sera in the lepromatous than in the tuberculoid and borderline leprosy groups.


Distribution of axonal transport blockade by acute intraocular pressure elevation in the primate optic nerve head. We studied the degree of axonal transport blockade in various areas of the optic nerve head with acute intraocular pressure (IOP) elevation in 19 squirrel monkey eyes. When IOP was raised to 20 to 50 mm. Hg for 7 hr., mild axonal transport blockade occurred in each area of the disk, most prominently in nerve fiber bundles of the superior pole. With 7 hr. IOP elevations between 50 and 90 mm. Hg, a somewhat greater degree of transport blockade occurred throughout the nerve head, although again the superior and inferior poles were somewhat more affected. The distribution of short-term transport blockade over the entire nerve head corresponds to the diffuse damage of acute glaucoma, but the pattern hints at the preference for damage near the poles of the disk seen in chronic glaucoma. However, before these results can be fully evaluated, further information is needed on axonal pathways through the optic nerve head and on the relationship between transport obstruction and ganglion cell death.


Synthesis of alpha-fetoprotein and some other serum proteins in testicular tumors. Serum levels of alpha-fetoprotein were determined in 33 patients with testicular germ cell tumors and were normal in 11 patients with seminoma and in one patient with matured teratoma; high levels were observed in 19 of 21 patients with embryonal carcinoma, teratocarcinoma, or a mixed type of these germ cell tumors. Tissues from the testicular germ cell tumors were cultivated with 14C-labeled leucine. After incubation, the culture media were subjected to immunoelectrophoretic and autoradiographic analyses. The results were: (i) Radioactive alpha-fetoprotein, albumin, transferrin, and alpha1-globulin appeared in the culture media of embryonal carcinomas obtained from two infants. (ii) Radioactive albumin and alpha1-globulin appeared in the culture media of a mixed type tumor metastasized from testis to retroperitoneal region. (iii) No such radioactive proteins appeared in the culture media of primary seminomas.


A modified allochrome procedure for demonstrating mycobacteria in tissue sections. A modified allochrome staining procedure is presented as being the most reliable and sensitive method for demonstrating mycobacteria in tissue sections. The technic is as follows: Deparaffinize formalin fixed sections, oxidize in 10% periodic acid for 24 hours, differentiate in 1% HCl-70% ethanol, stain in Weigert's iron hematoxylin nuclear stain, and counterstain in picro-methyl glue. Mycobacteria stained brilliant red in contrast with the allochrome-stained background tissues, and apparently otherwise chromophobic bacilli are demonstrated.


Hormone response of benign hyperplastic prostate tissue in organ culture. Glandular benign prostatic hyperplasia specimens from 10 patients were culutured for 4 days in media containing either insulin alone, insulin plus estradiol, insulin plus testosterone, insulin plus human placental lactogen, insulin plus human placental lactogen plus testosterone, or insulin plus human growth hormone plus testosterone. Growth of the specimens was defined by pulsing 3H-thymidine on day 3, terminating on day 4, and determination of acid-insoluble radioactivity by scintillation counting. The response of each specimen was related to its base line response to insulin media. Six of nine specimens were stimulated 80 per cent or more by insulin plus placental lactogen plus testosterone, but only one of seven was stimulated by insulin plus testosterone. Four of 10 were stimulated by insulin plus estradiol. Histologic and autoradiographic results indicated that all growth occurred in the epithelium.


Pharmacokinetic studies of a transplantable murine renal adenocarcinoma. An animal model to investigate new therapeutic approaches for the treatment of renal adenocarcinoma has been further studied. This model is based on a transplantable murine renal adenocarcinoma whose growth follows Gompertzian kinetics, relates to tumor RNA and DNA content, and also correlates with the rate of tumor DNA synthesis. This model in the current study was also evaluated for the ability of various therapeutic agents to inhibit tumor DNA synthesis. Such tests may be valuable for the preclinical screening of potentially useful drugs and may provide insight into fundamental aspects of tumor control. In this study, CCNU, BCNU, and adriamycin were potent inhibitors of tumor DNA synthesis whereas cytosine arabinoside, bleomycin, and cyclophosphamide were not. These observations were confirmed by autoradiography and correlated with other experimental end points of tumor therapy such as tumor weight and animal survival. This preclinical screening model is an effective and helpful means whereby new drugs and drug combinations can be tested for potential use in human renal cell carcinoma.


Weight, stroma to gland ratio, and mitotic activity of the human hyperplastic prostate. The growth of the hyperplastic prostate of 12 patients was studied with an in vitro 3H-thymidine labeling method. The prostates were weighed and the stroma to gland ratio was analyzed. The mitotic activity of the glandular epithelium averaged 0.36 per cent (+/- 0.06 SE). No mitotic activity was found in the stromal tissue. There was a positive correlation between the weight of the prostate and the labeling Index of the glandular epithelium. Thus, the prostatic hyperplasia of these patients appeared to be purely glandular in form. The stroma to gland ratio was 70 to 80 per cent. It was found that most of the glandular follicles were mitotically quiescent, and that the mitotic activity was concentrated in a few of the follices. The results indicate that the growth of the hyperplastic prostate is caused both by the mitotic activity (hyperplasia) of the glandular epithelium and by the stromal hypertrophy. Thus, in vitro assay of the mitotic activity of the prostatic biopsies can be used for the estimation of the weight of the hyperplastic prostate. The possible role of the stroma in regulation of the mitotic activity of the glandular epithelium has been discussed.


[Contagious ecthyma with virus demonstration in a negative staining method]. The diagnosis of contagious ecthyma was presumed by history and clinical appearance in a 33-year-old farmer. In electron miscroscopical preparations viral particles were found by the negative staining method. It is proposed to use this method for rapid diagnosis of suspected cases. In old lesions it is more difficult to demonstrate viral particles than in fresh lesions. Differential diagnosis and the position of Orf-virus in the system of viruses are briefly discussed.


Standardization of the post-irradiation method to eliminate primary fluorescence in cytofluorometry. Thorough irradiation of specimen with the strong excitation light after fluorescent Feulgen staining destroys the primary fluorescence in the background with stabilization of specific fluorescence of pararosaniline (post-irradiation method). An apparatus to perform effective post-irradiation was developed and irradiation condition for DNA cytofluorometry on a pararosaniline Feulgen stained smear was standardized. After 10 h irradiation on this condition, the primary fluorescente in the background is almost completely cleared away. Proportionality between amount of fluorescence and DNA content is perfectly preserved and standard deviation in each class of ploidy becomes to be less than 5% of the mean DNA value. The standardized post-irradiation enables us to obtain reproducibly the same values of fluorescence on Feulgen stained nuclei of the same cell population in different smears even if the staining conditions in Schiff's solution might be different to some extent.


Model system studies of staining procedures for lysine and arginine residues. Using polyacrylamide films containg poly-lysine, polyarginine and DNA as test models, a variety of reportedly specific staining procedures have been examine. Contrary to published observations, mixtures of fast green and eosin Y show no specific staining of either lysine or arginine. Both amino-acids bind eosin from the mixture more strongly than fast green. Arginine apparently has a greater affinity for this eosin than has lysine which contradicts previous reports that lysine will be stained by eosin arginine will stain with fast green, if proteins containing both amino-acids are stained with dye mixture. In films containing lysine and/or arginine picric acid is shown to bind specifically to the arginine. The picric acidarginine complex resists disruption in 0.004 M borate buffer which is a solvent used for subsequent staining of lysine residues with bromophenol blue. Picric acid may also be used as a hydrolysant and substitute for hydrocholoric acid in a Feulgen-like procedure which stains DNA to the same level as the classiclal hydrochloric acid based procedure while also staining arginine present.


Electrocardiographic interpretation of thiobarbiturate-induced dysrhthymias in dogs. The cardiac dysrhythmia occuring most commonly during intravenous administration of thiamylal sodium is ventricular bigeminy (ventricular premature depolarization coupled to the preceding sinus beat). Electrocardiographic tracings obtained during thiamylal-induced dyshythmias must be interpreted accurately if an accurate prognosis is to be give. Ventricular bigeminy seemed to originate in the ventricle, distal to the bundle of His. When ventricular premature depolarization was late in diastole, occuring simultaneiously with the next sinus-conducted impulse, a fusion beat resulted. The resultant ventricular bigeminy appeared as: (1) alternating ventricular premature dipolarizations, (2) an electrical alternans, and (3) alternating preexcitation syndrome. This dysrhythmia was associated with palpable alternating strong and weak frmoral arterial pulse. The likelihood that ventricular dysrhythmias will be caused by thiamylal is closely related to dose, concentration, and rate of administration. In a 23-month period, clinical occurrence of ventricular bigeminy during induction of anesthesia with thiamylal was approximately 4%.


Training autistic children to urinate in the toilet through operant conditioning techniques. The purpose of this study was to evaluate the use of operant conditioning techniques to toilet train children in an autism ward of a hospital for developmentally disturbed children. Five profoundly retarded males with clear clinical manifestations of autism were selected as subjects. Records of the urination behavior of these subjects were kept during a baseline period and throughout the application of procedures. Appropriate urination behavior was immediately followed by positive reinforcers, such as candy, verbal praise, and physical affection. Inappropriate urination behavior was immediately followed by negative reinforces, verbal as well as physical. The results of this study show that operant conditioning techniques can be used to change the urination behavior of profoundly retarded autistic children even where other methods have failed. Factors requiring further investigation for their possible impact on the effectiveness of these procedures in toilet training autistic children are also discussed.


Genetic basis of Vi antigen expression in Salmonella paratyphi C. Analysis of hybrids formed in a cross between a Salmonella paratyphi C Hfr and an S. typhimurium recipient indicated that the structural genetic determinants of the S. paratyphi C Vi antigen are located closely adjacent to the mel determinant, between this marker and purA. A similar location was indicated for the structural genetic determinants of the S. typhi Vi antigen (the viaB locus) by the results of a mating in which a hybrid S. typhimurium Hfr bearing the S. typhi viaB determinants was used to transfer these genes to an S. typhimurium recipient. Mating experiments with a Vi-antigen-expressing S. typhi Hfr and an S. typhimurium hybrid recipient expressing the Vi antigen of S. paratyphi C yielded no recombinants in which loss of Vi antigen expression occurred, indicating that the chromosomal locus occupied by the genetic determinants of the S. paratyphi C Vi antigen is the same one at which, in S. typhi, the viaB genes reside. Introduction of a mutant S. typhi viaA gene into an S. typhimurium hybrid expressing the Vi antigen, as the consequence of prior receipt of the S. paratyphi C viaB determinants, resulted in that hybrid's loss of Vi antigen expression, demonstrating that the viaA determinant plays a role in Vi antigen expression in S. paratyphi C, as well as in S. typhi. Although the percentages of coinheritance of the viaB and mel determinants in the mating experiments suggested that their linkage is sufficiently close to allow cotransduction by P22, attempts to accomplish this with lysates prepared on S. typhimurium hybrids expressing either S. typhi or S. paratyphi C viaB determinants were not successful.


Color key in projection graphics.

Color key in projection graphics. The authors describe a philosophical approach to projection graphics, and a cookbook procedural approach to the use of the 3M Color Key process, with variations on recommended methods. The philosophy is Mr. Goldenberg's, the recipes Mr. Ross's.


Detergent solubilization, purification, and separation of specificities of HLA antigens from a cultured human lymphoblastoid line, RPMI 4265. HLA antigens have been purified to homogeneity after detergent solubilization from RPMI 4265, a human lymphoblastoid line. The inhibition of cytotoxicity assay for HLA antigen was modified, using preincubation with bovine serum albumin of antigen samples containing detergent to prevent lysis of target cells by detergent. Solubilization was tested with many types of detergents. A polyethyleneglycol oleyl ether nonionic detergent mixture, Brij 99:Brij 97 (2:1) was selected for solubilization, since it selectively solubilized HLA antigens, had a low absorbance at 280 nm and was uncharded. HLA antigens were then purified by Lens culinaris lectin affinity chromatography and Bio-Gel A-5m filtration. The antigen specifity HLA-A2 was separated from specificities HLA-B7,12 by isoelectric focusing. Purified HLA antigens contained a subunit of Mr=44,000 with NH2-terminal glycine, and a subunit of Mr=12,000, beta2-microglobulin, with NH2-terminal isoleucine.


Five alpha-D-galactopyranosyl-binding isolectins from Bandeiraea simplicifolia seeds. The alpha-D-galactopyranosyl-binding lectin previously purified from Bandeiraea simplicifolia seeds (Hayes, C.H., and Goldstein, I.J. (1974) J. Biol. Chem. 249, 1904) is shown to consist of five isolectins separable on polyacrylamide gel electrophoresis at pH 9.5. The isolectins are tetrameric structures composed of various combinations of two different glycoprotein subunits designated A and B. The A and B subunits appear to be immunochemically indistinguishable against rabbit antisera prepared from the isolectin mixture. The A subunit contains no methionine, whereas the B subunit contains 1 residue. The subunits migrate differently on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and, although each subunit contains 1 residue of cysteine, they react differently toward 5,5'-dithiobis(2-nitrobenzoic acid). The carbohydrate binding specificity of the two subunits differs significantly: the A subunit exhibits a primary specificity for alpha-D-GalNAcp but also reacts with alpha-D-Galp units, whereas the B subunit shows a sharp specificity toward alpha-D-Galp residues. The differences in carbohydrate binding specificity were exploited in separating the isolectins. B. simplicifolia I isolectins (A4) and (A3B) were purified on a Bio-Gel melibionate column, and (A2B2), (AB3), and (B4) were separated on a column of insolubilized blood group A substance.


Human beta-globin messenger RNA. III. Nucleotide sequences derived from complementary DNA. Sequences of human beta-globin mRNA were determined by analysis of complementary DNA. beta-mRNA was transcribed into double-stranded cDNA by RNA-dependent DNA polymerase. cDNA was cut by restriction endonucleases and the fragments were terminally labeled by means of polynucleotide kinase and [gamma-32P]ATP. After purification, fragments were degraded by snake venom phosphodiesterase. Alternatively single-stranded [32P]cDNA was prepared by transcription in the presence of [alpha-32P]dCTP and actinomycin D; the product was digested by endonuclease IV and degraded by snake venom phosphodiesterase. cDNA tracts obtained by both labeling methods enabled us to construct a sequence for the translated and 3'-terminal untranslated regions of human beta-mRNA.


Immunological and biochemical studies of collagen type transition during in vitro chrondrogenesis of chick limb mesodermal cells. This work describes an approach to monitor chondrogenesis of stage-24 chick limb mesodermal cells in vitro by analyzing the onset of type II collagen synthesis with carboxymethyl-cellulose chromatography, immunofluorescence, and radioimmunoassay. This procedure allowed specific and quantitative determination of chondrocytes in the presence of fibroblasts and myoblasts, both of which synthesize type I collagen. Chondrogenesis was studied in high-density cell preparations on tissue culture plastic dishes and on agar base. It was found that stage-24 limb mesenchymal cells initially synthesized only type I collagen. With the onset of chondrogenesis, a gradual transition to type II collagen synthesis was observed. In cell aggregates formed over agar, type II collagen synthesis started after 1 day in culture and reached levels of 80-90 percent of the total collagen synthesis at 6-8 days. At that time, the cells in the center of the aggregates had acquired the typical chondrocyte phenotype and stained only with type II collagen antibodies, whereas the peripheral cells had developed into a "perichondrium" and stained with type I and type II collagen antibodies. On plastic dishes plated with 5 X 10(6) cells per 35mm dish, cartilage nodules developed after 4-6 days, but the type II collagen synthesis only reached levels of 10-20 percent of the total collagen. The majority of the cells differentiated into fibroblasts and myoblasts and synthesized type I collagen. These studies demonstrate that analysis of cell specific types of collagen provides a useful method for detailing the specific events in the differentiation of mesenchymal cells in vitro.


Immunological and ultrastructural studies of neurofilaments isolated from rat peripheral nerve. Neurofilaments were isolated from desheathed and minced segments of rat peripheral nerve by osmotic shock into 0.01 M Tris-HCI buffer, pH 7.2. Freshly isolated neurofilaments were observed to undergo disassembly by progressive fragmentation upon exposure of dilute tissue extracts to this buffer. Low- and high-speed centrifugations of these tissue extracts separated membranous and particulate constituents and produced a progressive enrichment of 68,000-dalton polypeptide band in successive supernates, as determined by analyses of soluble proteins by SDS-polyacrylamide electrophoresis. The final high-speed supernatant fractions (S3) of nerve extracts, which were predominantly composed of 68,000-dalton polypeptide, were used to raise a specific experimental antisera in rabbits. Utilizing techniques of immune electron microscopy, experimental rabbit antisear was shown to contain antibodies against neurofilaments. Intact neurofilaments isolated from rat nerves and attached to carbon-coated grids became decorated when exposed to experimental rabbit antisera or purified gamma globulin (IgG) derivatives. The decoration of neurofilaments closely resembled the IgG coating seen in immune electron microscopy. Antibody absorption techniques were used to identify the biochemical constituency of neurofilamentous antigenic determinants. The decoration of neurofilament by experimental IgG was not altered by additions of tubulin or bovine serum albumin, but was prevented by additions of S3 fractions as well as the 68,000-dalton polypeptide of this fraction which was eluted and recovered from polyacrylamide gels. These findings are indicative that a 68,000-dalton polypeptide is a constituent subunit of rat peripheral nerve neurofilaments.


Temporal expression of membrane antigens during mouse spermatogenesis. The temporal expression of cell surface antigens during mammalian spermatogenesis has been investigated using isolated populations of mouse germ cells. Spermatogenic cells at advanced stages of differentiation, including pachytene primary spermatocytes, round spermatids, and residual bodies of Regaud and mature spermatozoa, contain common antigenic membrane components which are not detected before the pachytene stage of the first meiotic prophase. These surface constituents are not detected on isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, or leptotene and zygotene primary spermatocytes. These results have been demonstrated by immunofluorescence microscopy, by complement-mediated cytotoxicity, and by quantitative measurements of immunoglobulin (Ig) receptors on the plasma membrane of all cell populations examined. The cell surface antigens detected on germ cells are not found on mouse thymocytes, erythrocytes, or peripheral blood lymphocytes as determined by immunofluorescence and by cytotoxicity assays. Furthermore, absorption of antisera with kidney and liver tissue does not reduce the reactivity of the antibody preparations with spermatogenic cells, indicating that these antigenic determinants are specific to germ cells. This represents the first direct evidence for the ordered temporal appearance of plasma membrane antigens specific to particular classes of mouse spermatogenic cells. It appears that at late meiotic prophase, coincident with the production of pachytene primary spermatocytes, a variety of new components are inserted into the surface membranes of developing germ cells. The further identification and biochemical characterization of these constituents should facilitate an understanding of mammalian spermatogenesis at the molecular level.


The behavior of alpha2-plasmin inhibitor in fibrinolytic states. Human plasma alpha2-plasmin inhibitor in fibrinolytic states was studied using immunochemical methods and radioiodinated plasminogen. The concentration and activity of plasma alpha2-plasmin inhibitor decreased when urokinase was added to plasma in vitro or infused intravenously in man. The decrease was associated with the appearance of plasmin-alpha2-plasmin inhibitor complex which subsequently disappeared from the circulation in a short time. A decrease of other major inhibitors, such as alpha2-macroglobulin and alpha1-antitrypsin, was not observed when the amount of urokinase added or infused was relatively small, and conversion of plasminogen to plasmin was not extensive. The formation of plasmin-alpha2-macroglobulin complex was observed only when plasma plasminogen was activated with a larger amount of urokinase, and after most of the alpha2-plasmin inhibitor was consumed by forming complexes with plasmin. The formation of plasmin-alpha1-antitrypsin complex was not observed even in the highly activated plasma unless exogenous plasmin was added to the plasma. alpha2-Plasmin inhibitor was the only inhibitor of which the concentration in plasma was significantly decreased in patients with disseminated intravascular coagulation and fibrinolysis among the major plasmin inhibitors in plasma. The most reactive inhibitor for regulating plasma fibrinolysis very likely is alpha2-plasmin inhibitor.


Trimellitic anhydride-induced airway syndromes: clinical and immunologic studies. This report describes a spectrum of respiratory symptoms in workers exposed to trimellitic anhydride (TMA), a biologically reactive chemical used in the plastics industry. Fourteen workers who had worked on a unit which synthesized TMA were evaluated by clinical and immunologic methods. Respiratory syndromes induced by TMA inhalation included asthma and rhinitis of the immediate type, late onset asthma with systemic symptoms, and airway irritation. TMA was shown to couple rapidly to human serum albumin, forming an immunoreactive hapten-protein complex. The workers' immunologic reactivity to this complex could be quantitated and correlated with the three respiratory syndromes. The asthma-rhinitis syndrome was mediated by IgE antibody specific for the TMA hapten. The syndrome of late onset asthma with systemic symptomes was accompanied by elevated levels of TMA-specific IgG antibody. Rheumatoid factor in high titer was found in one worker with IgE-mediated asthma and in two workers with asthma of late onset. Lymphocyte reactivity of TMA-HSA was demonstrated in three workers representative of the three clinical syndromes. Leukocyte histamine release was demonstrated to TMA-HSA in one worker with high levels of IgE antibody specific for TMA-HSA who had severe symptoms of acute rhinitis and asthma.


Conditions affecting the immunosuppressive properties of human alpha-fetoprotein. The hypothesis that alpha-fetoprotein (AFP) is immunosuppressive in vitro was tested with immunoabsorbent purified human cord AFP in human lymphocyte cultures. Albumins were purified identically from cord plasma and pooled adult plasma. No preparations were suppressive in phytohemagglutinin-stimulated cultures. Both AFP and human cord albumin (HCA) produced 50% suppression of allogeneic lymphocyte cultures at 50 to 100 microgram/ml final concentrations, whereas adult and commercial albumins were not suppressive. When AFP was eluted from the immunoabsorbent column in 0.15 M NaCl rather than the conventional 0.5 M NaCl, activity was greatly diminished or lost, but could be restored by dialysis against 0.5 M KCl. Previously, inactive lots of HCA also demonstrated the same phenomenon. It appears, therefore, that the immunosuppressive activity of AFP (and albumin) may depend both on its source and the procedure by which it is isolated. Whether analogous conditions occur in vivo is unknown at this time.


Precipitation of radiolabeled antigen-antibody complexes with protein A-containing Staphylococcus aureus. The feasibility of using protein A-containing Staphylococcus aureus to measure antibodies in sera from several mammalian species was studied. A variety of unrelated radiolabeled antigens were tested, including components of bovine serum, DNA, and bacterial and tumor-associated extracts. The use of S. aureus was found to be a reliable way to detect and measure the primary interactions between many of the antigens and antibodies tested. Results were equivalent under many circumstances to those obtained with the ammonium sulfate and heterologous anti-immunoglobulin methoods. However, some of the limitations noted were that certain antigens bound directly to S. aureus and that all classes of human immunoglobulins tested, in particular IgG3 and IgA1, were not precipitated by S. aureus. If these limitations are taken into consideration, the use of S. aureus can be of value in studying immunochemical reactions with other antigens.


Nature of the antigenic complex recognized by T lymphocytes. IV. Inhibition of antigen-specific T cell proliferation by antibodies to stimulator macrophage Ia antigens. We have previously demonstrated that nonimmune guinea pig T lymphocytes could be specifically sensitized with TNP-modified allogeneic macrophages after eliminating the alloreactive T cells with bromodeoxyuridine (BUdR) and light treatment. This procedure allowed the unique opportunity to use anti-Ia sera directed against the Ia antigens of only the stimulator macrophages or responder T cells to determine against which cell type anti-Ia would block TNP-specific stimulation. It was found that the TNP-specific DNA synthetic response of BUdR and light-treated T cells stimulated with TNP-modified allogeneic macrophages was totally eliminated by anti-Ia sera directed solely against the allogeneic stimulator macrophage. In contrast, anti-Ia sera directed only against the responder T cells had no effect on their response to TNP-modified allogeneic macrophages. These findings indicate that macrophage Ia antigens are required for efficient T cell-macrophage interactions and raise the possibility that T cell Ia antigens may not be required for collaboration with macrophages. This latter possibility was substantiated by experiments in which we show that treating T cells with anti-Ia sera and complement to remove the Ia-positive cells either before or after priming, or both, had no effect on their ability to be primed and restimulated with TNP-modified macrophages.


Enhancement of weak mixed lymphocyte-type reactions by hydrophilic polymers. Two hydrophilic polymers, dextran and polyethylene glycol, were found to enhance in vitro MLR-type reactions which are weak or nonexistent in normal culture media. BALB/c and C57BL/6 thymocytes were cultured for 4 days in Eagle's MEM + 10% FCS, with or without the x-irradiated lymphomas LSTRA AND EL-4, respectively. By thymidine incorporation no stimulation was observed with EL-4 in plain culture whereas occasionally a very small stimulation was observed with LSTRA. Four percent dextran (w/v, m.w. greater than or equal to 40,000) improves stimulation (E/C) by about 2-fold. The effect of polyethyleneglycol m.w. 6,000 (PEG-6) is much more pronounced. At the optimal concentration of 4 to 5%, PEG-6 causes an increase of 5- to 20-fold in the stimulation index. The PEG-6 effect can also be observed microscopically by the appearance of numerous blasts clustered around the tumour cells. PEG-6 by itself has little effect on  thymocytes in the absence of tumor cells. In the case of one-way allogeneic MLR under suboptimal ratios, dextrans (m.w. greater than or equal to 40,000) enhance the reaction dramatically. We speculate that these effects are occurring via a change in the solvent (such as solvent exclusion) in a way which enhances immunologic interactions under conditions which are tolerable to cells in vitro.


Immune suppression with supraoptimal doses of antigen in contact sensitivity. I. Demonstration of suppressor cells and their sensitivity to cyclophosphamide. Immunologic suppression was induced in a mouse model of contact sensitization to DNFB by using supraoptimal doses of antigen. In these studies, in vivo measurement of ear swelling as an indication of immunologic responsiveness correlated well with measurement of in vitro antigen-induced cell proliferation. This unresponsiveness was specific, since supraoptimal doses of DNFB did not interfere with the development of contact sensitivity to another contactant, oxazolone. The decrease in responsiveness is a form of active suppression, as lymphoid cells from supraoptimally sensitized donors transferred suppression to normal recipients. Furthermore, pretreatment with cyclophosphamide (Cy) reversed the suppression seen in supraoptimally sensitized animals but had no effect on the optimal sensitization regimen. These results indicate that supraoptimal doses of contactants can activate suppressor cells and that precursors of these cells are sensitive to Cy. Such suppressors regenerate within 7 to 14 days after Cy treatment. The ability of Cy pretreatment to affect supraoptimal sensitization without affecting optimal sensitization confirms other reports indicating that the observed results of Cy treatment depend critically upon the dose of antigen used.


Immunologic tolerance to heterologous immunoglobulin: its relation to in vitro filtration by macrophages. The cellular and molecular basis for the difference in ability of BCG to induce tolerance in BALB/c and DBA/2 mice has been examined by in vitro biofiltration. It was found that incubation with the adherent cells from BALB/c but not DBA/2 spleens could remove the material from BGG which inhibited tolerance induction in BALB/c mice. This material was shown to represent only a trace component in BGG, was present in only certain commercial batches of BGG, and was apparently unrelated to the presence of aggregates or endotoxin.


Evaluation of radioimmunoprecipitation for the detection of bovine leukemia virus infection in domestic cattle. The potential application of a recently developed radioimmunoprecipitation test for antibody directed against the major structural protein of bovine leukemia virus was evaluated for use in detection of BLV-infection in domestic cattle. This technique was found to be considerably more sensitive than serologic procedures currently being utilized for this purpose. Radioimmunoprecipitation was also shown to have distinct advantages as compared to hematologic criteria, such as specified by Bendixen's index, for identification of BLV-infected animals. By the use of radioimmunoprecipitation, high levels of antibody to BLV were demonstrated in sera of animals with confirmed adult lymphosarcoma, but not in animals with a less common sporadic form of the disease which occurs in calves.


Neonatally induced tolerance to HGG: duration in B cells and absence of specific suppressor cells. A specific, long lasting, tolerant state to human gamma-globulin (HCG) was established in neonatal A/J mice. These suckling mice received the tolerogen in the colostrum of their mother who had been injected with DHGG. The tolerant state could not be accounted for by "factors" other than HGG in the colostrum. The duration of this tolerance in the intact animal and in the B cell population was 16 to 18 weeks. Naturally occuring nonspecific suppressor cells were evident but specific suppressor cells could not be demonstrated. These results are discussed in relation to possible mechanisms of the induction of tolerance to self.


Enhancement of the DNA synthetic response of antigen-primed lymph node cells by splenic promoter cells: characterization of the splenic promoter cells. Rabbits were immunized in the footpad with diphtheria toxoid in complete Freund's adjuvant. At various times after immunization, cells harvested from the spleen, the draining (immune) and the opposing (control) lymph nodes (LN) were assayed for their proliferative response to incorporate tritiated thymidine upon exposure to the priming antigen. Although the immune LN cells responded by a substantial incorporation of thymidine, cells from the control LN and the spleen either did not respond or responded feebly. An enhancement in the response of the immune LN cells was observed when they were cultured in the presence of nonresponsive spleen cells. Pretreatment of spleen cells with mitomycin C did not abolish the enhancement. This suggests that the target cells which respond to the antigen are derived from the immune LN whereas the promoter cells which enhance the response are present in the spleen. The removal of adhering cells by glass wool columns and of Ig-bearing cells by anti-Ig immunoabsorbent columns from the spleen did not reduce the enhancing capacity of the nonadhering cells. Conversely, the killing of splenic T cells by specific heterologous antiserum directed against rabbit thymus lymphocyte antigen abolished the enhancement. Thus, the promoter cell which facilitates the enhancement has been characterized as a nonadherent, splenic T cell.


Effects of alpha-fetoprotein on murine immune responses. II. Studies on rats. In vitro and in vivo experiments failed to identify a consistent "immunosuppressive" or "immunoregulatory" role for alpha-fetoprotein (AFP) in the rat. Both normal and AFP-rich sera inhibited Con A, PHA, and MLR proliferation responses of lymphocyte cultures in vitro equally well, in spite of up to 100,000-fold differences in AFP concentration. AFP-rich sera also had no effect on the antibody response of the rat in vivo. Purified AFP from amniotic fluid inhibited the PHA response at concentrations of 100 in chemical contexts is converted to microgram/ml. Purified albumin was also inhibitory, but at 1 log higher concentration. Purified AFP from tumor sera was not inhibitory at 100 in chemical contexts is converted to microgram/ml for Con A, PHA, or MLR reactions and only inhibited PHA stimulation at medium concentrations greater than 1000 in chemical contexts is converted to microgram/ml. The role of AFP as an immunosuppressive agent, particularly in reference to survival of mammalian fetuses, should be reconsidered.


Isolation of specific antibody under conditions of low ionic strength. Specific rabbit and goat antibodies to tobacco mosaic virus, bovine serum albumin and human gamma globulin have been isolated by acid dissociation from the antigen in the absence of salt. Maximum recovery of 96-100% of active antibody occurred at pH 2.8-2.9. The amount of antibody recovered within any given pH range was highly reproducible. The average antibody affinity was found to be correlated with the pth of dissociation and to decrease exponentially with the length of exposure to acid conditions.


In vitro induction of tumor-specific immunity. VI: analysis of specificity of immune response by cellular competitive inhibition: limitations and advantages of the technique. The cellular competitive inhibition 51Cr-release assay makes two distinct contributions to the in vitro study of cell-mediated immunity. It allows target cells which are not amenable to isotopic labelling to be investigated for their antigenic specificity, and it provides a means, complementary to the direct cytotoxicity assay, of estimating qualitative and quantitative differences in antigen expression on intact normal and neoplastic cells. Various parameters of a micro-51Cr-release inhibition assay have been studied, and it was found that the assay conditions markedly influenced both the sensitivity and specificity. It is concluded that optimal assay conditions for specificity include: 1) moderate levels of lysis on the linear part of the CL/T titration curve, 2) avoidance of prolonged assay times, and 3) low ratios of blocker to target cells. When tumor cells with large cell volumes are used as competitive inhibitor (blocker) cells, non-specific blocking will occur; limits have been defined for this particular micro-inhibition assay which, in general, exclude these effects.