In vivo testing confirms a blunting of the human cell-mediated immune mechanism during space flight.

The cell-mediated immune (CMI) mechanism was evaluated in 10 space shuttle astronauts by measuring their delayed-type hypersensitivity response to seven common recall antigens. The Multitest CMI test system was used to administer antigens of tetanus, diphtheria, Streptococcus, Proteus, old tuberculin, Candida, and Trichophyton to the forearm 46 h before nominal mission termination; readings were conducted 2 h after landing. The mean number of reactions was reduced from 4.5 preflight to 3.0 inflight, and the mean reaction score was reduced from 21.4 to 13.7 mm inflight. The data presented suggest that the CMI system is still being degraded by space flight conditions on day 4 and that between day 5 and day 10, the depression maximizes and the system begins to adjust to the new conditions. The relation of these in vivo findings to previously reported in vitro results is discussed.     

In situ suppression of delayed-type hypersensitivity: another mechanism for sustaining the immune privilege of the anterior chamber.

Immunological rejection of a highly immunogenic, syngeneic tumour (UV5C25) in the anterior chamber (AC) of BALB/c mice was analysed. Hosts developed systemic, tumour-specific cytotoxic T lymphocyte (CTL) activity (P less than 0.001) as well as systemic, tumour-specific delayed-type hypersensitivity (DTH) (P less than 0.001). Histopathological features of tumour rejection were consistent with that of a CTL-mediated process [i.e., piecemeal necrosis of individual tumour cells by tumour-infiltrating lymphocytes (TIL)]. There was no evidence of ischaemic necrosis, perivascular cuffing, infarction, or vascular damage, as expected of a DTH-mediated process. In an effort to selectively eliminate CTL or DTH effector cells and hence alter the pattern of tumour rejection, mice were treated with anti-CD8 or anti-CD4 antibodies, respectively. Elimination of either cell population not only eliminated both systemic CTL and DTH activity to this tumour, but also resulted in progressive tumour growth. Analysis of TIL from untreated tumour-bearing hosts demonstrated tumour-specific cytolysis (P less than 0.01) as well as the presence of DTH effector cells (P less than 0.01). These results indicate that while both DTH and CTL effector cells are present in the AC, only the latter are active in tumour resolution in the AC; DTH effectors are active systemically, but suppressed locally. Further, these data also suggest the requirement of a CD8+ cell population for the development of a systemic DTH response to this tumour.     

Down-regulation of immune responses to inhaled antigen: studies on the mechanism of induced suppression

Repeated exposure of rats to an aerosol of ovalbumin (OVA) or its dinitrophenylated derivative (DNP-OVA) induced carrier-specific tolerance to subsequent challenge with the same haptenated antigen. Following parenteral challenge with DNP-OVA, both anti-DNP and anti-OVA IgE titres were reduced relative to controls, whereas anti-DNP responses following challenge with DNP-Ascaris were normal. Stimulation of tolerant rats with OVA, together with the polyclonal B-cell mitogen LPS, restored their capacity to respond to the antigen. In contrast to WAG rats, which have previously been shown to develop equivalent tolerance in the IgE an IgG antibody classes (Sedgwick & Holt, 1984), BN rats exposed to an OVA aerosol developed high serum titres of anti-OVA IgG. Following parenteral challenge with DNP-OVA, however, anti-DNP IgG responses in the BNs were markedly reduced relative to unexposed controls, while anti-OVA IgG titres were maintained at a high level. Further strain-dependent differences in T-cell function in tolerized rats appeared in in vivo assays of DTH reactivity and in in vitro antigen-driven lymphocyte proliferation. Both BN and WAG rats displayed diminished in vitro responses, whereas DTH reactions were only suppressed in the latter strain.     

In vivo macrophage suppression of delayed hypersensitivity in the guinea-pig.

The nature of the suppressive activity in the peritoneal exudate cells (PEC) of guinea-pigs immunized with dinitrophenyl bovine gamma globulin (DNP50-BGG) was investigated. A method was developed to isolate from the peritoneal exudate large numbers of macrophages. Using density gradient centrifugation on Percoll it was possible to obtain a population of cells which contained over 90% macrophages. This macrophage preparation was found to respond to lymphokine but to be incapable of passively transferring delayed hypersensitivity reactions. When these immune macrophages were transferred into antigen immunized animals, which had been pretreated with cyclophosphamide (CY), the skin reactions were suppressed to the same extent as when the total PEC was transferred. PEC from guinea-pigs immunized with ovalbumin in Freund's incomplete adjuvant did not suppress the skin reactions in CY-pretreated DNP50BGG immunized animals. However, in contrast, macrophages from these animals did suppress the skin reactions in the recipient guinea-pigs indicating that the macrophage suppression was not antigen specific.     

In vivo biocompatibility studies. V. In vivo leukocyte interactions with Biomer

A cage implant system was utilized to quantitatively and qualitatively characterize in vivo leukocyte interactions with cast Biomer. Scanning electron microscopy (SEM) in conjunction with cytochemical staining procedures were used to investigate the cellular events at the leukocyte/Biomer interface as well as in the inflammatory exudate over a 21-day implantation period. SEM was used to characterize leukocyte morphology on the Biomer surface and the cytochemical stains were used to differentially count leukocytes and to demonstrate intracellular alkaline and acid phosphatase activity. The results showed that the population density of leukocytes on the Biomer surface diminished with implantation time. The population density of multinucleated foreign body giant cells remained constant with time, while the numbers of nuclei per giant cell increased. The differential analysis revealed that macrophages preferentially adhered to the Biomer surface compared to other leukocytes in the exudate. The phagocytic capability of all adherent leukocytes, including giant cells, decreased with time and this corresponded to changes in leukocyte morphology observed with SEM.     

Production and use of human monoclonal anti-D antibodies

Rhesus haemolytic disease of the newborn is a condition which can result in intrauterine or perinatal death. Although the passive administration of therapeutic anti-D post-partum is a most effective method for the prevention of this condition, there is currently a shortage of immune plasma for the preparation of the therapeutic anti-D immunoglobulin product. In addition the availability of anti-D for use in blood grouping has also been reduced. The advances made in recent years in the techniques for the production of human monoclonal antibodies raise the possibility that human monoclonal anti-D-based products may provide solutions to both of these problems. There are now a number of reports of the production of stable cell lines secreting high titre human anti-D. In this review we consider the various strategies used in the production of human monoclonal anti-D-secreting cell lines, the basic properties of these reagents and their potential usefulness in blood grouping, in therapy and as research tools.     

In vivo turnover studies of C-reactive protein.

The in vivo plasma clearance rate of the acute phase reactant C-reactive protein (CRP) was studied in mice and rats. The clearance rate of 125I-human CRP in mice and 125I-rat CRP in rats showed a T1/2 of approximately 4 h. The T1/2 was independent of circulating levels of CRP and was not affected by the presence of C-polysaccharide (CPS), a ligand to which CRP binds. However, in mice receiving sufficient CPS, more radioactivity localized to the spleen compared to mice receiving 125I-CRP only. 125I-CPS was rapidly cleared at the same rate by normal mice and by mice undergoing an acute phase response while rats cleared 125I-CPS more slowly despite having high circulating CRP concentrations. These findings suggest that CRP does not provide a mechanism for extremely rapid clearance of its ligands from the circulation, although the handling and subsequent fate of these ligands may be affected.     

In vitro studies of `antigenic competition': II. Reconstitution of the immune defect and the relationship between antigen-induced suppression and non-specific enhancement

The experiments described in this paper extend the observations of previous work in vitro demonstrating a decrease in the frequency of antigen-reactive units specific for horse RBC in spleen cells from mice primed with KLH. It was observed that the addition of lymphoid cells having T-cell function reconstituted the anti-HRBC response to normal values. Significant enhancement of the response beyond the normal values could be evoked using two dissimilar methods, (i) addition of allogeneic thymus cells, and (ii) restimulation in vitro with the priming antigen. The latter type enhancement was elicited by the addition of small doses of KLH to cultures of spleen cells from mice recently primed with KLH, including cultures otherwise demonstrating antigen-induced suppression. The stimulus required to enhance the response to HRBC in these cultures was specific for the priming antigen, KLH. These results are discussed in the light of current theories of `antigenic competition' and specific heterologous enhancement.     

In vivo suppression of T-dependent antibody responses by treatment with a monoclonal anti-L3T4 antibody

Minute amounts of the anti-L3T4 antibody designated GK1.5 were found to deeply suppress in vivo antibody responses to T-dependent antigens. Primary responses to sheep erythrocytes were completely inhibited even when GK1.5 was administered up to 6 days before or 4 days after the antigen. Secondary responses to potent immunogens like sheep erythrocytes or keyhole limpet hemocyanin were also completely abolished by a single injection of GK1.5 just before the boost. This treatment had no effect on T-independent reactions such as the polyclonal activation of B lymphocytes with lipopolysaccharide or the anti-2,4,6-trinitrophenyl (TNP) response elicited by injection of TNP-Ficoll.     

In vivo genotoxicity studies with p-benzoquinone dioxime.

P-Benzoquinone dioxime (BQD) appears to be a sex-specific rat carcinogen inducing tumours of the urinary bladder in female rats. The present paper shows that BQD is a direct-acting mutagen in Salmonella typhimurium TA98, confirming published data. In contrast to this in vitro data, negative results were obtained after oral administration of BQD to female rats in both the bone marrow micronucleus test and the in vivo liver UDS test. BQD did, however, induce a marked effect upon S-phase synthesis in the livers of female rats between 14 and 48 hr after a single oral dose of 250 mg/kg. A similar effect was also observed in the livers of male rats. There was no evidence of hepatotoxicity (in terms of elevated liver enzyme levels) after treatment of female rats with the compound indicating that the increase in cell proliferation was due to a direct mitogenic effect of BQD in this organ. Some liver mitogens have been found to be liver carcinogens; this does not appear to be the case for BQD. Nevertheless, the mitogenic activity of this compound might play a contributory role to the induction of bladder cancer in rats if it also acted as a mitogen in this tissue. Further studies are indicated, measuring genotoxicity and cell-proliferative activity in the bladder in order to further elucidate the mechanism of action of this compound as a rodent carcinogen.     

In vivo studies of cell-mediated and humoral immune responses to adenovirus 12-induxced tumour cells

Summary Mice immunized with 3 or 5 doses of cell-free extract from adenovirus 12-induced tumour cells were relatively immune to subsequent challenge with 5 × 10⁵ viable adenovirus 12-induced tumour cells. Using spleen cell transfer experiments, this immunity was shown to be cell mediated. Thus, subcutaneous inoculation with spleen cells from mice immunized with tumour extract together with tumour cells in a 151 ratio, respectively, inhibited tumour growth, as shown by a reduction in tumour incidence and the size of tumours, compared to the control mice inoculated with tumour cells and normal mouse spleen cells. When the tumour cells and spleen cells were given by different routes of inoculation, inhibition was not as marked as when the two were given together. Mice inoculated with spleen cells, from mice immune to challenge with live tumour cells (tumour immune mice) or spleen cells from mice bearing large transplantable tumours, were shown to be relatively immune to transplanted tumour cells. Transfer of serum from mice immunized with tumour extract to normal mice did not confer any immunity to transplanted tumour cell challenge; tumour growth was neither retarded or enhanced, compared to control mice given normal CBA mouse serum. Transfer of serum from mice immune to tumour cell challenge also did not affect the growth of tumours. However, transfer of serum from tumour-bearing mice resulted in an apparent earlier development of tumours in two distinct experiments; however, in neither experiment was the difference statistically significant compared to the tumour incidence in control mice.     

The clearance kinetics of autologous RhD-positive erythrocytes coated ex vivo with novel recombinant and monoclonal anti-D antibodies

Anti-D is given routinely to pregnant RhD-negative women to prevent haemolytic disease of the fetus and newborn. To overcome the potential drawbacks associated with plasma-derived products, monoclonal and recombinant forms of anti-D have been developed. The ability of two such antibodies, BRAD-3/5 monoclonal anti-D IgG (MAD) and rBRAD-3/5 recombinant anti-D IgG (RAD), to clear RhD-positive erythrocytes from the circulation was compared using a dual radiolabelling technique. Six RhD-positive males received autologous erythrocytes radiolabelled with (99m)Tc and (51)Cr and coated ex vivo with MAD and RAD. Blood samples were collected up to 1 h following intravenous injection, and percentage dose of radioactivity in the samples determined. Three different levels of coating were used on three separate occasions. No significant differences between MAD and RAD were observed in the initial clearance rate constant at any dose level. The log[activity]-time clearance plots were curved, showing a reduction in the clearance rate constant with time. This reduction was more marked for RAD than for MAD. The results support a dynamic model for the clearance of antibody-coated erythrocytes that may have wider relevance for the therapeutic use of antibodies.     

In vivo protection against S. mansoni infection by monoclonal antibodies

A monoclonal antibody of the IgG1 class (27.21), that was previously shown to be cytotoxic in vitro to the schistosomula, conferred partial protection in vivo against challenge infection with S. mansoni. Monoclonal antibodies of the IgE class (54.10) were not effective in these experiments, but were capable of causing specific degranulation of basophilic cells upon contact with the young larva (3-6 h). The relevance of the IgE antibodies to protection observed in immunized mice is discussed.     

In vivo metabolism of a monoclonal IgG cryoglobulin

The metabolism of a monoclonal IgG cryoglobulin was studied in a patient with primary idiopathic cryoglobulinaemia under two different clinical conditions. Early in the illness, cryoprotein levels were diminished to 770 mg% by plasmapheresis and plasma disappearance assayed while serum levels of cryoprotein increased to 1100 mg%, i.e. the study was performed during a period of unstable cryoprotein synthesis. A second study was performed 1 year later when serum levels of cryoglobulin were stable at 1500 mg% which allowed a synthetic rate of 64 mg/kg of body weight per day to be computed. This was the upper limit of normal IgG synthesis. The final slopes of the plasma disappearance curves were nearly identical for both studies, indicating that the same fractional percentage of the cryoglobulin serum pool was degraded regardless of its serum level. T1/2 of 15·5 and 17·0 days were obtained when plasma levels were unstable and stable, respectively.

For comparative purposes, the plasma disappearance of pooled normal IgG, IgG₃ myeloma proteins and a highly aggregated IgG₃ myeloma were also studied when cryoprotein levels were 1500 mg%. Normal IgG synthesis was suppressed and only 8·5 mg/kg body weight per day, but its plasma disappearance curve showed a final slope nearly identical to those for the cryoprotein, indicating that the ability to catabolize normal IgG was unimpaired. IgG₃ myelomas were cleared at their normal accelerated rate, while aggregated IgG₃ was totally cleared from the circulation within 2 days, indicating that the patient was able to catabolize circulating immune complexes.

As controls, the catabolism of cryoprotein was shown to be identical to that of pooled normal IgG in two volunteers.

The data support the concept that the build up of circulating IgG cryoprotein in the patient studied was due to an increase in cryoprotein synthesis and not to a lack of ability to catabolize it.

     

In vivo studies of individual mucous glands in the frog.

Individual mucous glands in the toe web were studied in curarized decerebrate frogs using vital microscopy in combination with still or motion photomicrography. By changing the focus position to different levels various structures in the gland could be identified and their changes during glandular activation studied. The first visible effect of nerve stimulation was a contraction of the myoepithelium and probably also structural changes of the secretory epithelium resulting in a narrowing of the glandular lumen. Following this, tricuspid valve opened and secretion was ejected. The latency and time course of the contractile response to nerve stimulation were determined and the influence of the number of stimuli on the duration of the contraction and relaxation phases was analyzed. Comparisons were made with reflex activation of the gland as well as with neurohormonal stimulation. The myoepithelial contraction was found to be under adrenergic control. Of the smooth-muscle stimulants tested only Substance P induced contractions. The time course of the ionic outflow from the toe web was determined by conductance measurements in the fluid surrounding the web and compared with the visually observed phenomena. The initial outflow was concomitant with the phasic myoepithelial contraction but a continued secretion could also be observed and recorded from glands kept in a steady state of contraction by iterative nerve stimulation. The functions of the toe web glands were found to be critically dependent on a maintained circulation in the surrounding capillary network.     

Flow cytometric analysis of 68 monoclonal anti-D reagents

68 monoclonal IgG anti-D were assessed for quantitative and qualitative binding to D variant red cells using a flow cytometric indirect immunofluorescence test. One antibody failed to bind to normal RhD pos. cells, but reacted weakly with D cat VI and D Cat VII cells. Quantitatively, binding varied approximately twenty-fold between different MAbs and different D variants. D cat III cells gave the lowest level of binding when compared with the D pos. controls. Qualitatively, R₁^VIr, R₂^VIr, R₁^VIIr and DFR samples failed to react with various MAbs regardless of the levels of binding of these MAbs to the D pos. controls, thus supporting the idea of missing epitopes in such variants.