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Antibodies against HTLV-III, neopterin levels in blood and urine, TH/TS ratio and hepatitis marker were determined in 34 clinically symptom-free persons known to be intravenous drug abusers. 15 persons were positive in the ELISA and Western-blot tests. There was a strong reaction to protein p24 compared with that to protein p41. In 12 of 14 persons who were antibody-positive the neopterin level in morning urine was elevated; an abnormal TH/TS ratio was present in nine of 13 persons. In future, determination of neopterin and of antibodies against certain proteins of HTLV-III may make it possible to provide a simple way of prognosticating on the course of an HTLV-III infection.

Aging is a multi-facetted process, but the deterioration of the immune function seems to play a central role in this context. Paradoxically, immune reactivity against exogenous antigens declines during aging while autoimmune reactivity increases. One of the aims of our investigations on the function of the senescent immune system was to define immune parameters of 'normal' aging, i.e. those not dependent on underlying diseases. Specifically, our interest was focused on the possible role of an altered lipid metabolism of cells of the immune system during aging. The known decrease of plasma membrane fluidity of lymphoid cells and monocytes in higher age may be one of the factors responsible for the nonoptimal functioning of the immune system. This property, in turn, seems to be based on an altered lipid metabolism. Specifically, we have evidence that the finely tuned balance between the transport of cholesterol to and from lymphoid cells via the environment and the intracellular cholesterol biosynthesis seems to be disturbed with increasing age. This conclusion is drawn from experiments where low-density lipoprotein (LDL) and high-density lipoprotein (HDL) receptor activity is assessed using fluorescently labeled lipoproteins in fluorescence-activated cell sorter (FACS) analyses. LDL receptor uptake is unexpectedly increased in the elderly, but LDL receptor regulation, and serum LDL composition itself seem to be normal. Preliminary data point out the possibility that the efflux of cholesterol via HDL may be insufficient. Attempts to modulate plasma membrane fluidity by means of the phospholipid mixture 'active lipid 721' (AL 721) showed that this drug, in contrast to literature reports, is not a 'membrane fluidizer' but rather exerts this effect as a nutrient for lymphocytes and monocytes.

Lymphomatous goiter (struma lymphomatosa) first described by Hashimoto in 1912(1), was shown to be an autoimmune disorder 30years ago(2). In the sameyear an autoimmune thryoiditis was first induced experimentally by immunization with autologous thyroid extract and complete Freund's adjuvant(3). Among the many insights gainedfrom the study of such experimentally induced thyroiditis(3-5) is the notion that susceptibility to it is under a genetic control which influences both the reactivity of the immune systems and the properties of the thyroid gland(7). This is supported by the appearance of autoimmune thyroiditis in genetically restricted animal populations. Here Georg Wick and his colleagues review recent studies of the Obese strain (OS) of chickens and compare the pathogenesis of theirspontaneous autoimmune thyroiditis with that of Hashimoto's disease.

Aims Decreased night-time plasma levels of melatonin were recently reported in patients with coronary artery disease, and it was postulated that melatonin production may be impaired, due to a lack of synthesizing enzymes. However, since artefacts possibly influencing the release pattern were not taken into account, this interpretation was strongly criticized. We therefore carefully investigated night-time melatonin production in patients with coronary artery disease using an appropriate experimental approach. Furthermore, we examined the effect of beta-blockers, a frequently used drug in coronary artery disease therapy. Methods and Results Forty-eight male patients with angiographically documented severe coronary artery disease, 24 of them taking beta-blockers daily in therapeutic dosages, were included. Eighteen age-matched men, with no evidence of coronary sclerosis, served as controls. To determine melatonin production, 6-sulfatoxymelatonin (aMT6s) was measured radioimmunologically from overnight urine. Urinary aMT6s concentration was significantly decreased in patients, and beta-blocker treatment did not further suppress melatonin production. Conclusions The data obtained using this investigative approach provide clearcut evidence that melatonin production in patients with coronary artery disease is decreased. Whether a decreased melatonin level may be a predisposing factor for coronary artery disease, or whether the occurrence of coronary artery disease decreases melatonin synthesis remains to be determined.

AIM Until now no serological markers were available towards the diagnosis of squamous-cell carcinomas of the head and neck (HNSCC) particularly in the detection of posttherapeutic recurrent diseases and metastases. Previous reports described patients with malignant diseases of various tissues exhibiting a characteristic and highly significant alteration in the subclass composition of serum IgG, consisting of a reduction in %IgG1 and an increase of %IgG2. In this study we present for the first time results of this IgG-shift in patients suffering from HNSCC. PATIENTS AND METHODS A total of 111 patients was investigated at our clinic, all suffering from primary, histopathologically verified squamous cell carcinomas of the head and neck. These patients were investigated as to %Ig G1/IgG2 prior to any treatment. A second group consisted of 35 patients with local recurrences, 15 patients with distant metastases and 27 patients without tumour at the time of investigation, who were in observation for 1 to 5 years after primary treatment (T0). Thirdly, a total of 33 patients was included who were afflicted with a variety of benign diseases of the head and neck, such as chronic rhinosinusitis, chronic tonsillitis and also lateral or median cysts of the neck. Data of the three groups were compared with those of 174 healthy volunteer controls. 5 ml blood were taken from a forearm vein and the quantitation of subclasses IgG1, IgG2 and total IgG was performed by affinity chromatography. The single values obtained with all experimental groups and healthy controls showed normal distribution for primary cancer patients versus healthy control. Accordingly, significant differences between mean values were calculated with the two-sided Students t-test, and cut-off values were calculated as the arithmetic means of mean values obtained from the groups to be compared. Diagnostic sensitivities and specificities were defined as percentages of patients with %IgG1 smaller, and of healthy controls with %IgG1 greater than the cut-off value. RESULTS We found a highly significant alteration in the subclass composition of serum IgG, consisting of a reduction in %IgG1 and an increase in %IgG2 in our HNSCC groups. The present data suggest the changes in %IgG1 and %IgG2 as a useful serological tumour marker to detect primary or recurrent and/or metastatic squamous-cell carcinomas of the head and neck. CONCLUSION The shift in %IgG1/%IgG2 exhibited diagnostic sensitivities and specificities comparable to, or--particularly at early tumour stages--by far higher than conventional serological tumour markers. Whereas conventional serological markers directly correspond to tumourogenically derived products, the shift in %IgG1/IgG2 represents an indirect marker, consisting of a change of the host's immune system due to the presence of malignant tumours.

For several years, our group has been studying thein vivorole of adrenergic and cholinergic mechanisms in the immune-neuroendocrine dialogue in the rat model. The main results of these studies can be summarized as follows: (1) exogenous or endogenous catecholamines suppress PBL functions through alpha-2-receptor-mediated mechanisms, lymphocytes of the spleen are resistant to adrenergicin vivostimulation, (2) direct or indirect cholinergic treatment leads to enhancedex vivofunctions of splenic and thymic lymphocytes leaving PBL unaffected, (3) cholinergic pathways play a critical role in the “talking back” of the immune system to the brain, (4) acetylcholine inhibits apoptosis of thymocytes possibly via direct effects on thymic epithelial cells, and may thereby influence T-cell maturation, (5) lymphocytes of the various immunological compartments were found to be equipped with the key enzymes for the synthesis of both acetylcholine and norepinephrine, and to secrete these neurotransmitters in culture supernatants

Immunoglobulin G (IgG) of many species contains 'labile' disulfide bonds (SS*), which within 24 h undergo a disulfide exchange with dithionitrobenzoic acid (NBSSBN). Aims of the present study were to detect directly this type of SS* groups by means of 14C-labelled NBSSBN, and to investigate its possible presence in other serum proteins. NBSSBN reacts during the first 30 min of incubation with free SH groups, and thereafter with the sulfur atoms of SS* groups. The latter reaction reaches equilibrium after 24 h. The total of thionitrobenzoate residues (NBS) bound to IgG is called 'sigma S' and represents both SH and SS*. The results can be summarized as follows: (1) The measurement of the binding of 14C-NBSSBN gave identical sigma S values with IgG from humans and mice, as compared to the detection with the unlabelled reagent, which is based on the photometric determination of liberated NBS anions; whereas with IgG from rats some differences were observed which were ascribed to different batches of animals investigated; (2) experiments with electrophoretically separated serum proteins revealed only the gamma-globulins strongly binding 14C-NBSSBN in addition to the 30 min reaction, which indicates that SS* is confined to the gamma-globulin fraction; and (3) the significant decrease of sigma S in association with malignant tumors in man and animal models, which was previously described to be due to a specific alteration of the IgG subclass pattern, was likewise detected with the radiometrical method. Previous studies have identified SS* as one of the two inter-heavy disulfide bridges in IgG1, and possible implications of this group in specific functions of IgG1 are discussed.

Immunoenhancing in vivo effects of beta-adrenergic blockers have been previously ascribed to a reduced beta-receptor-mediated immunosuppression. In the present study using a whole blood stimulation assay, the effects of a five-day treatment with the purified (R)- or (S)-isomer of propranolol (3 x 40 mg/day) on the polyclonal in vitro responsiveness of peripheral blood lymphocytes (PBL) of normothyroid and hyperthyroid persons were assessed. It is shown that both isomers likewise exhibit a significant enhancing effect on the proliferative response of PBL to T and B cell mitogens, which strongly argues for nonspecific effects of propranolol to be responsible rather than a specific beta-adrenergic receptor blockade.

Besides the pineal gland, melatonin is reported to be produced in a number of extrapineal sites, where it could act as an intracellular mediator or paracrine signal in addition to its endocrine effects. In view of the suggested immunoregulatory role of melatonin, we compared lymphoid organs and several other tissues of the rat for their potential to synthesize melatonin. Using the reverse transcription-polymerase chain reaction (RT-PCR) method, we determined the tissue-specific expression of mRNAs encoding two key enzymes of the melatonin biosynthesis: serotonin-N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). The minimal number of PCR cycles required to obtain a positive signal served as a measure for the abundance of a given mRNA. NAT and HIOMT mRNAs were detected in all tested tissues at high numbers of PCR cycles (40 and 45, respectively). At 35 cycles, only gut, testis, spinal cord, raphe nuclei, stomach fundus and striatum yielded positive signals for both enzymes. In conclusion, the presence of NAT and HIOMT mRNAs in a wide range of tissues corroborates and extends the notion of extrapineal melatonin synthesis. Comparatively low levels of the HIOMT messages in lymphoid organs, however, indicate a limited significance of melatonin synthesis within the immune system.

Norepinephrine stimulates growth of Escherichia coli, Yersinia enterocolitica, and Pseudomonas aeruginosa in serum-supplemented media, and in vivo increases in norepinephrine may be important in the pathogenesis of sepsis by gram-negative bacteria. Because salmonellosis often is associated with stress, the effects of norepinephrine on in vitro growth, and in vivo pathogenicity of the swine pathogen Salmonella choleraesuis were investigated. When RPMI 1640 with and without pig serum was inoculated with fewer than 100 S. choleraesuis/ml and incubated overnight, bacterial numbers were 10(4) to 10(6) lower in RPMI containing serum. Norepinephrine restored bacterial growth in RPMI with serum to normal levels, but it did not increase growth in serum-free RPMI. Similar results were obtained with SAPI, a nutrient-poor medium previously used to study the effect of norepinephrine on growth of gram-negative bacteria. Conditioned media were produced by growing S. choleraesuis in RPMI containing serum with and without norepinephrine and filter sterilizing. Conditioned medium produced with norepinephrine stimulated growth of S. choleraesuis but not E. coli, whereas conditioned medium produced without norepinephrine stimulated growth of both bacteria. To determine the in vivo effects of norepinephrine, rats were implanted with tablets that secrete norepinephrine for 20 to 24 hours or with identical tablets without norepinephrine and infected intraperitoneally with graded doses of S. choleraesuis. The LD-50 of S. choleraesuis was the same in both groups, and norepinephrine did not affect the carrier rate at 30 days after infection. We concluded that although norepinephrine stimulates in vitro growth of S. choleraesuis in serum-based media, the increase in norepinephrine levels in the present in vivo system was probably not sufficient to influence the pathogenesis of S. choleraesuis infection.

Plasma levels of interleukin-1beta (IL-1beta), IL-2, soluble IL-2 receptor (sIL-2R), IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha), and the p60 soluble TNF receptor (sTNFR) were repeatedly determined by enzyme-linked immunosorbent assays (ELISA) in 35 patients with different subtypes of juvenile rheumatoid arthritis (JRA) during an observation period of up to 36 months. The data were related to conventional inflammatory parameters and disease activity. Patients with systemic disease showed the most pronounced elevations of plasma cytokines, followed by polyarticular and pauciarticular JRA. Soluble receptors sIL-2R and sTNFR were consistently elevated in patients of all JRA subtypes and indicated disease activity even in patients with normal C-reactive protein (CRP). In contrast, the determination of IL-1beta, IL-2, IL-8, and TNF-alpha revealed strikingly different individual profiles in patients of the same clinical subtype of JRA and irrespective of disease activity. It is concluded that the determination of sIL-2R and sTNFR may be relevant for monitoring JRA, as they indicate disease activity also in cases with unaltered conventional inflammatory parameters. The different individual cytokine profiles of patients within identical subtypes of disease suggest JRA to be even more heterogeneous than hitherto assumed. The data should be considered in attempts to develop anticytokine strategies in the therapy of JRA.

Until now no serological markers were available towards the diagnosis of squamous-cell carcinomas of the head and neck (HNSCC) particularly in the detection of posttherapeutic recurrent diseases and metastases. Previous reports described patients with malignant diseases of various tissues exhibiting a characteristic and highly significant alteration in the subclass composition of serum IgG, consisting of a reduction in %IgG1 and an increase of %IgG2. In this study we present for the first time results of this IgG-shift in patients suffering from HNSCC.A total of 111 patients was investigated at our clinic, all suffering from primary, histopathologically verified squamous cell carcinomas of the head and neck. These patients were investigated as to %Ig G1/IgG2 prior to any treatment. A second group consisted of 35 patients with local recurrences, 15 patients with distant metastases and 27 patients without tumour at the time of investigation, who were in observation for 1 to 5 years after primary treatment (T0). Thirdly, a total of 33 patients was included who were afflicted with a variety of benign diseases of the head and neck, such as chronic rhinosinusitis, chronic tonsillitis and also lateral or median cysts of the neck. Data of the three groups were compared with those of 174 healthy volunteer controls. 5 ml blood were taken from a forearm vein and the quantitation of subclasses IgG1, IgG2 and total IgG was performed by affinity chromatography. The single values obtained with all experimental groups and healthy controls showed normal distribution for primary cancer patients versus healthy control. Accordingly, significant differences between mean values were calculated with the two-sided Students t-test, and cut-off values were calculated as the arithmetic means of mean values obtained from the groups to be compared. Diagnostic sensitivities and specificities were defined as percentages of patients with %IgG1 smaller, and of healthy controls with %IgG1 greater than the cut-off value.We found a highly significant alteration in the subclass composition of serum IgG, consisting of a reduction in %IgG1 and an increase in %IgG2 in our HNSCC groups. The present data suggest the changes in %IgG1 and %IgG2 as a useful serological tumour marker to detect primary or recurrent and/or metastatic squamous-cell carcinomas of the head and neck.The shift in %IgG1/%IgG2 exhibited diagnostic sensitivities and specificities comparable to, or--particularly at early tumour stages--by far higher than conventional serological tumour markers. Whereas conventional serological markers directly correspond to tumourogenically derived products, the shift in %IgG1/IgG2 represents an indirect marker, consisting of a change of the host's immune system due to the presence of malignant tumours.

First indications that the pineal gland may be involved in endocrine immunomodulation came from early reports on anti-tumor effects of pineal extracts in animals and humans. In the meantime, evidence has accumulated suggesting that melatonin, the major endocrine product of the pineal gland-as a well preserved molecule during evolution-is indeed involved in the feedback between neuroendocrine and immune functions. At present we are beginning to understand the mechanisms of action by which melatonin affects cellular functions, and from the variety of possible direct and indirect interactions it appears that melatonin may play a complex physiological role in neuroimmunomodulation. In this article we present a critical review of the numerous reports on the influence of melatonin on immune functions and discuss the possible underlying molecular pathways. In addition, a short comment is given on the current public discussion as to the clinical value of melatonin.

Immunoglobulin G (IgG) of many species contains 'labile' disulfide bonds (SS*), which within 24 h undergo a disulfide exchange with dithionitrobenzoic acid (NBSSBN). Aims of the present study were to detect directly this type of SS* groups by means of 14C-labelled NBSSBN, and to investigate its possible presence in other serum proteins. NBSSBN reacts during the first 30 min of incubation with free SH groups, and thereafter with the sulfur atoms of SS* groups. The latter reaction reaches equilibrium after 24 h. The total of thionitrobenzoate residues (NBS) bound to IgG is called 'sigma S' and represents both SH and SS*. The results can be summarized as follows: (1) The measurement of the binding of 14C-NBSSBN gave identical sigma S values with IgG from humans and mice, as compared to the detection with the unlabelled reagent, which is based on the photometric determination of liberated NBS anions; whereas with IgG from rats some differences were observed which were ascribed to different batches of animals investigated; (2) experiments with electrophoretically separated serum proteins revealed only the gamma-globulins strongly binding 14C-NBSSBN in addition to the 30 min reaction, which indicates that SS* is confined to the gamma-globulin fraction; and (3) the significant decrease of sigma S in association with malignant tumors in man and animal models, which was previously described to be due to a specific alteration of the IgG subclass pattern, was likewise detected with the radiometrical method. Previous studies have identified SS* as one of the two inter-heavy disulfide bridges in IgG1, and possible implications of this group in specific functions of IgG1 are discussed.

In recent studies using a cold pressor test we could show that former opiate addicts are persistently less pain-sensitive than healthy controls, indicating a neurophysiologic dysfunction in these patients. In the present study we addressed the issue of whether this dysfunction was caused by the drug abuse or already existed prior to the heroin addiction, and whether it is restricted to pain sensitivity or affects somatosensory or nociceptive sensitivity in general. After validating the method we obtained retrospective ratings for the pain, cold and warmth sensitivity for the time before addiction, during addiction and during detoxification. Ex-addicts perceive themselves less pain- and cold-sensitive than healthy controls, and no difference was detectable between the pre-addiction and the rehabilitation ratings, although nociceptive sensitivity is highly increased during detoxification. Warmth sensitivity was not different to healthy controls and was not affected by drug withdrawal. Our findings suggest that a decreased nociceptive sensitivity may already precede opiate addiction pointing to physiological dysfunctions in heroin pre-addicts.

The concept of an extrinsic regulation of the immune system through neuroendocrine signals is well established, as is the fact that the immune system in turn informs the brain about contacts with antigens via “immunotransmitters”, i.e. cytokines and/or hormones with central effects [1]. All these data that have accumulated during the last twenty years have contributed to the vision of the immune system as “the sixth sense” [2]. While there is certainly still more work needed to define the physiology of this concept in all details, strong evidence has been obtained that the immune-neuroendocrine dialogue is of relevance for the homeostasis of the immune response, as defects in the activation of the hypothalamo-pituitary-adrenal (HPA) axis by immune signals were found to be associated with and/or to predispose to spontaneously occurring [3] and experimentally induced autoimmune diseases in animal models [4, 5], and there is evidence that the same is true also in humans [6]. A large body of more recent literature data strongly suggests that this dialogue involves not only the hypothalamus, but several other brain areas, notably the structures of the “Limbic System” (for review see [7]).

The cholinergic system takes part in the immune-neuroendocrine integration. Lymphocytes have been found to express muscarinic and nicotinic cholinergic receptors, as well as acetylcholine-esterase on their surface, and cholinergic agents modulate immune functions in vitro and in vivo. In the present study we provide evidence that purified organ resident and circulating lymphocytes, as well as various lymphoid cell lines derived from different species, exhibit choline-acetyltransferase activity and, therefore, have the potential to produce the neurotransmitter acetylcholine.

The purpose of this article is to review recent experimental data from our laboratory on immune-neuroendocrine communications, whereby the following findings will be discussed: (i) Experiments using an experimental stress model in rats revealed different doses of a defined stressor to exert opposite effects on the in vitro reactivity of peripheral blood lymphocytes (PBL). Stress effects are also dependent on the tissue origin of lymphocytes, and they seem to be exclusively mediated by adrenal hormones. (ii) The balance between the sympathetic and parasympathetic nervous system may be critical in extrinsic immunoregulation: Experimentally induced hypercatecholaminemia in rats seem to protect lymphocytes from immunosuppressing effects of other, endogenous stress hormones, but causes suppression of PBL activation, if beta-receptors are blocked at the same time. Chronic cholinergic stimuli exert enhancing effects on the cells of the thymus. (iii) A defect in the immune-neuroendocrine crosstalk may contribute to the occurrence of forbidden immune reactions, as has been shown in spontaneous and experimentally induced autoimmune diseases in animals models. Recent data indicate that the parasympathetic nervous system is involved in the transmission of immune messages to the central nervous system.

Sigma S is a measure of the disulfide bonds and free thiol groups of serum immunoglobulin (Ig) G, as determined by the reaction with dithionitrobenzoate. Significant decreases of sigma S previously were detected in malignant compared with benign diseases of various organs. This study shows the application of sigma S for the diagnosis of breast cancer. The following results were obtained. First, 132 patients with benign breast diseases showed a sigma S of 1.48 +/- 0.29 (standard deviation) per mole IgG; this was not different from 1.51 +/- 0.36 found in 182 controls. In contrast, IgG from 198 patients with primary breast carcinoma of all four stages (tumor-node-metastasis system) gave a sigma S of 1.22 +/- 0.29, a significant (P less than 0.0001) decrease of sigma S from benign to malignant breast disease. Second, sigma S values of single Stages I, II, III, and IV, were 1.27 (n = 59), 1.23 (n = 83), 1.19 (n = 35), and 1.10 (n = 21), respectively, each significantly different from sigma S in benign disease and showing a decreasing trend with increasing tumor progress. Differences were significant between Stages I and IV (P less than 0.025) and II and IV (P less than 0.05). Third, 63% of Stage I breast carcinoma patients had sigma S values below a critical threshold of 1.38. This serum positivity rose to 90% in Stage IV. These values exceeded those reported with other tumor markers. The overall power of sigma S to distinguish between benign and malignant breast disease had a specificity of 61% and a sensitivity of 78%. Early stages (I and II) of breast cancer could be distinguished from benign diseases with 64% specificity and 69% sensitivity. Advanced Stage IV could be discriminated from early Stages I and II with 55% specificity and 71% sensitivity. Thus, the analysis of sigma S may significantly contribute to the surveillance of patients with breast cancer.

In-vitro evidence suggests that DNA methylation may be involved in the development of forbidden immune responses that can result in autoimmune disease. In the present study we examined in-vivo effects of 5-azacytidine (5-azaC), a substance that inhibits DNA methylation, on the immune system and the occurrence of a spontaneous autoimmune disease in the chicken model. We found that (1) treatment of young normal chickens with 1.0 mg/kg 5-azaC on 7 consecutive days caused a rapid degeneration of the central lymphoid organs thymus and bursa; (2) this regimen with 5-azaC apparently inhibited B cell maturation, as the frequency of cytoplasmic Ig+ plasma cells in the bone marrow was found to be significantly reduced, whereas the total number of bone marrow cells was unchanged; and (3) a chronic low-dose (0.5 and 1.0 mg/kg) application of 5-azaC through 6 weeks was found to significantly enhance the spontaneous autoimmune thyroiditis in newly hatched chickens of the Cornell C strain, as determined by anti-thyroglobulin autoantibody titres and histological analysis of thyroid gland infiltration. The possible implications of these data for the generation of pathogenic autoimmune responses are discussed.

Prior investigations showed that acutely diseased patients with an aspergillus sinusitis manifested immune dysfunctions in respect of both T and B lymphocytes. In contrast to patients with nonmycotic sinusitis reduced in vitro and in vivo responsiveness was observed. The aim of this study, carried out after removal of the fungus ball and endoscopic surgery in clinically healthy patients, was to ascertain whether this reduced responsiveness was to be regarded as the effect or cause of an Aspergillus fumigatus infection. Two years later, the in vivo response to recall antigens was normal in both groups of patients, whereas the response to mitogens (ConA, PHA and PWM) was still decreased in the aspergillus sinusitis groups. The data suggest that the reduced immune response is a consequence of the Aspergillus fumigatus infection. Depressed skin reactivity is only present during acute infection, while proliferative capacity, as measured in the "whole blood stimulation" assay is depressed for a long time after healing the acute infection.

The binding parameters of muscarinic antagonists in intact rat thymocytes were determined from competitive binding experiments with 3H-N-methylscopolamine (3H-NMS). The muscarinic antagonists inhibited binding of 3H-NMS in a dose-dependent manner. Non-linear regression analysis of the displacement curves indicated the presence of two affinity states for the muscarinic compounds. The beta-blocking agents R-propranolol, S-propranolol and alprenolol inhibited the binding of 3H-NMS to one, low affinity binding site on rat thymic lymphocytes. The ganglionic blocking substance hexamethonium did not affect the binding of 3H-NMS. The results indicate the presence of two binding sites for the radioligand on rat thymocytes, of which only one is specific for muscarinic receptor antagonists.

High concentrations of unsaturated fatty acids are known to cause hemolysis. At low concentrations, however, unsaturated cis fatty acids have been found to protect erythrocytes against hypotonic hemolysis. In the present experiments we examined the effect of oleic (18:1), linoleic (18:2), linolenic (18:3), and elaidic (18:1) acid on the osmotic fragility of chicken and sheep erythrocytes, which markedly differ in their resistance to osmotic rupture. The results are summarized as follows: (A) The phenomenon of stabilization was observed in both species alike. (B) Interaction of cells with the fatty acids under isotonic conditions led to a persistent stabilization, i.e., the cells remained more resistant against osmolysis even after several washings. (C) Oleic and elaidic acid protected against osmotic rupture with a high degree of specificity. Linoleic and linolenic acid were much less protective. Thus, this effect appears to be specific for one double bond. (D) Contrary to the unsaturated fatty acids with cis configuration, elaidic acid with the trans configuration showed no biphasic behaviour, and even at the highest concentrations applied no hemolysis was observed.

In contrast to systemic autoimmunity, spontaneous autoimmune thyroiditis of Obese strain (OS) chickens is associated with a marked T cell hyperreactivity in vitro , i.e. an increased proliferation and interleukin 2 (IL 2) secretion in response to Concanavalin A (ConA). In the present study we report an enhanced capacity of OS peripheral lymphoid cells (splenocytes and peripheral blood lymphocytes, PBL) to adsorb fluorescein isothiocyante (FITC) labelled ConA, but not phytohemagglutinin (PHA). However, the elevated ConA binding cannot be a prerequisite for in vitro ConA hyperreactivity as OS thymocytes are normal with respect to ConA binding but nonetheless exhibit elevated responses to this mitogen. Moreover, ConA binding does not correlate with the frequency of cells able to express IL 2 receptors upon short term ConA stimulation. The percentage of ConA activatable cells was found to be increased in OS- PBL as compared to normal control PBL, but was unaltered in OS splenocytes. This finding points to a further mechanism of T cell hyperreactivity in OS chicks in addition to the previously reported defects in nonspecific immunosuppression. Finally, enumeration of cells in the S phase revealed that enhanced proliferation of OS T lymphocytes was not restricted to the in vitro response to ConA and phytohemagglutinin (PHA) but also occurs in vivo .

Monoclonal antibody INN-CH 16 recognizes a surface determinant exclusively present on activated chicken T lymphocytes. The present experiments suggest that this chicken activated T lymphocyte antigen (CATLA) represents the chicken IL-2 receptor or an associated structure, as (i) the kinetics of CATLA expression during T cell activation are analogous to those described for the mammalian receptor for IL-2, (ii) the IL-2 dependent proliferation of mitogen prestimulated chicken lymphocytes is competitively inhibited by INN-CH 16, and (iii) pretreatment of T lymphoblasts with INN-CH 16 drastically reduces their capacity to absorb IL-2 activity from supernatants of mitogen activated chicken lymphocytes.

Conjunctivitis follicularis is characterized by the appearance of cellular infiltrates consisting of lymphocytes, eosinophils and mast cells; this has led to the assumption of an allergic etiology in many cases. In a first attempt to prove this hypothesis three cases of conjunctivitis follicularis were investigated using the following immunological methods: (1) determination of immunoglobulins in the lacrimal fluid by means of quantitative precipitation (IgG, IgA, IgM), and by a direct radioimmunoassay for IgE; (2) direct immunofluorescence test on cryostat sections of biopsy specimens of diseased conjunctiva to detect immunoglobulin-producing plasma cells. According to initial results the infiltrated conjunctiva contains considerable numbers of plasma cells producing mainly IgA, and IgM. Plasma cells positively stained with anti-IgG and notably with anti-IgE were only rarely found. These findings, which were also supported by the quantitative Ig determinations in the lacrimal fluids, tend to rule out an allergic, IgE-mediated pathomechanism in these cases.

The Obese strain (OS) of chickens, which is afflicted with Hashimoto-like spontaneous autoimmune thyroiditis (SAT), displays elevated T cell proliferation, interleukin (IL)2 production and IL2 receptor expression upon mitogen stimulation, and defects in the neuroendocrine control of the immune system including elevated corticosteroid-binding globulin (CBG) and a deficient increase of serum corticosterone (CN) upon cytokine injection. Recently this strain has further been shown to harbor retrovirus-related sequences (endogenous virus no. 22, ev22) absent in healthy control strains. To determine the number of genes responsible for SAT-associated immunodysregulation and to unravel possible ev22 associations, we analyzed the above immune and endocrine parameters in F1 hybrids and backcrosses of the autoimmune OS B15B15 with healthy inbred CB B12B12 chickens. OS-like T cell hyperproliferation and IL2 hypersecretion in response to both concanavalin A and phytohemagglutinin were transmitted as autosomal dominant traits and co-segregated in backcross animals. In vivo hyporesponse of the OS to the corticosterone-inducing effect of cytokine preparations was inherited dominantly and the elevated CBG serum levels recessively. None of these traits appeared to be major histocompatibility complex (MHC) linked. However, while T cell abnormalities and elevated CBG serum levels were not associated with the autosomal ev22 locus, in vivo hyporesponsiveness to glucocortocoid-inducing cytokines co-segregated with this OS-specific provirus. These results add to the concept of SAT as a polyetiological and plurigenetic disease and do not support our previous hypothesis that T cell hyperreactivity and immunoendocrine dysfunction might be functionally related.

In about 10% of patients operated on a chronic sinusitis, an aspergilloma is found in the paranasal sinus. To detect possible underlying immunodeficiencies, patients with aspergilloma were subjected to an immunological screening programme. The data were compared with those of patients suffering from non-mycotic chronic sinusitis and healthy controls. Totale lymphocyte counts and immunological levels were normal in both groups of sinusitis. Leukocyte subset analyses by membrane fluorescence revealed a significant decrease of CD11+ cells, i.e. macrophages/monocytes and NK cells, in both types of sinusitis. Furthermore, a markedly enhanced frequency of CD25+-cells, i.e. IL 2-receptor bearing cells, was observed in patients with aspergilloma. Peripheral blood lymphocytes of both groups of patients showed a significant reduction in the proliferative response to both T and B-cell mitogens, the values for the mitogens ConA and PWM being significantly lower in aspergilloma patients than in those with non-mycotic sinusitis. This lack of lymphocyte stimulation in the aspergilloma group was also manifest in skin tests to recall antigens. These first data suggest an immunodeficiency in association with chronic sinusitis caused by Aspergillus fumigatus. Further studies are needed to clarify if this defect is cause or result of the mycotic infection.

The exocytotic exposure of antigens of chromaffin granule membranes was studied with chromaffin cells isolated from bovine adrenal medulla. Antigens on the cell surface were visualized by indirect membrane immunofluorescence employing antisera against glycoprotein III and dopamine beta-hydroxylase. With unstimulated cells, only weak immunofluorescence on the cell surface was observed, whereas stimulated cells (with carbachol or Ba2+) exhibited much stronger reactions. In all cases the staining appeared as dots and patches. To quantitatively prove these observations, we analyzed the immunostained cells using a fluorescence-activated cell sorter. After stimulation, the average fluorescence intensity of the cell population was enhanced. This increase correlated with the degree of catecholamine secretion. The fluorescence intensity of stimulated cells varied over a broad range indicating that individual cells reacted variably to the secretagogues. When stimulated cells were incubated at 37 degrees C for up to 45 min after stimulation, a decrease of membrane immunofluorescence approaching that of unstimulated control cells was observed. Apparently, the membranes of chromaffin granules, which had been incorporated into the plasma membrane, were retrieved by a specific and relatively fast process. This retrieval of the antigen from the cell surface was blocked by sodium azide, but not influenced by colchicine, cytochalasin B, and trifluoperazine. The quantitative methods established in this paper should prove useful for further study of the kinetics of the exo-endocytotic cycle in secretory tissues.

Thyroglobulin-binding lymphoid cells were identified in the spleen of Obese strain (DS) chickens by their capacity to form rosettes with thyroglobulin-coated chicken red blood cells. The nature of these cells was studied in inhibition experiments using turkey anti-chicken bursa or thymus cell sera and rabbit antisera specific for chicken Ig, gamma, mu, alpha, Fabgamma or Fcgamma. Spleen cells actively synthesizing surface receptors for thyroglobulin were identified as B cells and the receptors found to be complete IgM molecules. Normal T cells became thyroglobulin-rosette-forming cells via passive adsorption of thyroglobulin antibodies, a phenomenon which could be inhibited competitively by the addition of normal chicken serum to the incubation medium. Thyroglobulin antibodies passively adsorbed onto the surface of normal T cells also belong to the IgM class as verified both by inhibition experiments and studies employing IgM and IgG fractions of a high titered OS serum for the preincubation of the cell suspensions. Only preincubation with the IgM fraction of the anti-thyroglobulin antibodies resulted in the formation of significant numbers of passive rosette-forming cells.

Stabilization of red cells against hypotonic haemolysis by cis-trans isomeric free C18 fatty acids occurs with pronounced specificity which is strongly temperature-dependent, but in a distinctly different manner for the two configurational isomers. Oleic acid (cis-18:1) stabilizes very efficiently at 0 degrees C, even at the highest concentrations. Elaidic acid (trans-18:1) causes neither stabilization nor haemolysis at this temperature. At room temperature (23 degrees C), elaidic acid acquires the ability to protect, without turning haemolytic at high concentrations. At 37 degrees C elaidic acid also becomes haemolytic. The protecting effect of oleic acid at 0 degrees C is the result of a rapid reaction. The characteristic, temperature-dependent specificity of cis-trans isomeric C18 fatty acid interaction with the red cell membrane appears to be a general phenomenon, since it was observed alike with erythrocytes of different species.

The fluorescence kinetics of fluorescein isothiocyanate (FITC)-antibody conjugates during short (0.01 sec) laser excitations were analyzed for possible information about the immunological specificity of binding to antigenic substrates in direct immunofluorescence assays with antigen-coated Sephadex beads. First results of these investigations suggest marked differences in the bleaching characteristics of specifically and nonspecifically bound FITC conjugates: FITC-anti-bovine serum albumin (BSA) and FITC-anti-rabbit immunoglobulin (Ig) were found to fade significantly more slowly when bound to the respective homologous antigen (anti-BSA to BSA, and anti-rabbit Ig to rabbit Ig) as compared to nonspecifically adherent anti-BSA to rabbit Ig, and anti-rabbit Ig to BSA. Possible implications of these data for discrimination of nonspecific staining in immunofluorescence are discussed.

In general there exist two possibilities to obtain quantitatively information in immunohistochemical work: (1) Titration of an antibody of defined specificity and concentration on different antigenic substrates. (2) The measurement of the "specific signal" of the marker molecule used for the visualization of the specific fixation of antibody to the homologous antigen in histological preparations. Examples for both kinds of approach are given by recent data from quantitative immunofluorescence (IF) studies performed in this laboratory. In the second part of this talk some theoretical aspects of quantitative IF are discussed concerning the fluorescence properties of fluorescein isothiocyanate, the most widely used fluorochrome. Macrospectrofluorometric measurements revealed marked effects on the emission intensity of the composition of the embedding medium and the concentration of the dye. Furthermore, the conjugation to protein is shown to cause a considerable quenching of UV excited fluorescence, but not at excitation with visible blue (496 nm). A further critical point in quantitative IF is the illumination source. The great advantages of laser light excitation as compared to filtered light of the most widely used mercury arcs are discussed. Finally some experimental data concerning the recovery of already faded fluorescence as observed with short, repeated laser light pulses are mentioned. The practical significance of these theoretical data are stressed.

Peanut agglutinin (PNA) has turned out to be a potent tool to distinguish between cortical and medullary lymphocytes in the mouse thymus (1,2). In addition, more recent studies revealed that PNA may preferentially bind to cells acting as suppressors. PNA+ suppressor cells were detected in the fetal liver (3) and aging mouse spleen (4), as well as in the thymus of young adult animals (5). Furthermore, antigen-specific suppressors in the adult spleen were also characterized as PNA+ (6,7). Whereas the thymic and the antigen-specific splenic suppressor cells have been identified as T cells, the embryonic ones, and those appearing in aging were not eliminated by anti Thy 1.2 antibodies and complement (7 and unpublished observations). Hence, PNA seems to bind to suppressor cells of various membrane phenotypes. The possibility that PNA may enable identification and separation of various types of suppressor cells within the different lymphoid cell compartments thus seemed intriguing.

Excitation and emission properties of fluorescein derivatives were studied macrofluorometrically. Measurements were performed with solutions of various concentrations (0.07-100 microgram/ml) of free sodium fluorescein prepared from fluorescein diacetate (FDA), fluorescein isothiocyanate (FITC) and FITC bound to rabbit gamma-globulin. Both excitation and emission spectra as well as fluorescence intensities at constant excitation/emission wavelengths (496/515 nm) were recorded. The findings indicate that (1) FDA gives about twice the fluorescence intensity compared to equal concentrations of FITC. (2) The fluorescence properties of FITC upon excitation with blue light (lambda = 496 nm) are only slightly altered by the conjugation to rabbit gamma-globulin. (3) Considerable quenching due to conjugation could, however, be shown to occur upon UV excitation (lambda = 340 nm). (4) Fluorescence emission excited by visible blue light (496 nm) increases linearly to dye concentration in a range of 0.07-2.5 microgram/ml. Beginning at 5 microgram/ml (10-(5) M/1) all three compounds show a sharp decrease of fluorescence intensity with further increasing concentration. Practical aspects of these data for the immunofluorescence method are discussed.

It has been postulated that the establishment of acquired, neonatal immunologic tolerance is due to a "deficit" in interleukin 2 (IL-2). To test this hypothesis, chickens were made immunologically tolerant to both major and minor histocompatibility antigens by transplantation of skin grafts onto newly hatched recipients. In this study, we injected various doses of IL-2 and concanavalin A simultaneously with transplantation and in some cases, several days posttransplantation, and we failed to enhance graft rejection. These results may have practical importance in respect to the clinical use of recombinant IL-2. Injection of IL-2 in and around surviving skin grafts also failed to alter skin graft survival.