Autoantibody production in rabbits: V. Comparison of the autoantibody response after the injection of rat and rabbit liver and brain

The injection of rat liver and rat brain in Freund's complete adjuvant into rabbits led to the production of complement fixing autoantibodies against rabbit tissue. The autoantibody response following the injection of rabbit liver and brain was much smaller.

The autoantibody response to rat liver occurred even when Freund's incomplete adjuvant was used. The injection of rabbit liver in Freund's complete adjuvant into rabbits which had produced autoantibody after the injection of rat liver did not increase the level of autoantibody to rabbit liver.

In most, but not all, rabbits the natural antibody found in the sera taken before immunization was destroyed by heating the sera at 65°. After immunization with rat liver in Freund's complete adjuvant the levels of serum antibody to rabbit liver stable at 56° rose by day 5; however, autoantibody stable at 65° was not detectable until between days 7 and 14.

     

Validation of autoradiography for recognition of antigen-binding lymphocytes in blood and lymphoid tissues: quantitation and specificity of binding

Antigen-binding lymphocytes were recognized by their reaction with radioiodine labelled antigens such as flagellin and haemocyanin. Counts varied according to the antigen and species studied. For flagellin, counts in human blood of antigen-binding lymphocytes (mean ± 1 SD per 1000 lymphocytes) were 19·0±3·0, and in foetal thymus 18·2±5·0 and spleen 3·5±0·5. Results depended on contact time of cells with antigen, concentration of antigen, autoradiographic exposure, presence of natural antibody and antibody levels after immunization. Antigen-binding lymphocytes in blood were not antibody-producing cells. The specificity of the antigen-binding reaction was shown by exposing lymphocytes to 0·5 μg of two antigenically distinct flagellins; there was a 67–100% increase in the counts in contrast to the 20–45% increase on doubling the dose (0·5 μg to 1 μg) of flagellin from Salmonella adelaide. Cytophilic antibody as the cause of antigen binding was excluded.

The binding of flagellin to lymphocytes was prevented by anti-human IgM and light chain antisera, but not anti-human IgG sera. The binding of labelled flagellin was prevented by unlabelled flagellin but 100 times more was needed for blood lymphocytes than thymocytes. It is inferred that thymocytes, T cells, have considerably fewer receptors than most β lymphocytes detectable in blood.

Using standardized conditions, radiolabelled antigen binding provides a reproducible, immunologically specific and flexible technique allowing study of the nature and role of antigen-binding cells and cell surface receptors.

     

The metabolism of autologous IgM and 19S rheumatoid factor in rheumatoid arthritis

The turnover of autologous preparations of radio-iodine labelled IgM and 19S rheumatoid factor was studied and compared in patients with severe rheumatoid disease. The IgM was isolated by block electrophoresis and column chromatography. 19S rheumatoid factor was isolated by a combination of euglobulin precipitation, column chromatography, and absorption onto and acid-elution from solid aggregated IgG.

Ten studies were made in seven patients, five with IgM, and five with 19S rheumatoid factor. In two patients the turnovers of 19S rheumatoid factor and IgM were studied simultaneously.

The turnover of IgM was similar to that reported for normal subjects and patients with other diseases: fractional catabolic rate 0·14–0·18, plasma and whole body T_½ 3·7–6·5 days, with 65–77% intravascular localization. The absolute catabolic rate for IgM was elevated (8–60 mg/kg/day).

The turnover of 19S rheumatoid factor isolated from serum was comparable in fractional catabolic rate (0·15–0·19) plasma and whole body T_½ (3·9–6·0 days) and intravascular localization (62–88%). No evidence of rapid catabolism of the `immune' elimination type was obtained.

     

Lymph node localization and whole body distribution of radioiodinated encephalitogenic polypeptide in guinea-pigs

A bovine encephalitogenic polypeptide (BEP) labelled with radioiodide retained its capacity to induce experimental encephalomyelitis (EAE). Guinea-pigs were injected with ¹²⁵I BEP in Freund's complete adjuvant (FCA), to study changes in the architecture and the distribution of radioactivity in draining lymph nodes, and the amount of radioactivity in various organs.

After injection of BEP in FCA the lymph node rapidly enlarged. Within 48 hr there was depletion of lymphocytes, the enlarging lymphoid follicles had become confluent and there was proliferation of large `epithelioid' cells throughout the node. At 5 days the lymph node architecture was disorganized and lymph follicles with germinal centres could not be recognized; similar but less pronounced changes were present in regional nodes. By contrast, after injection of flagellin in FCA, there were numerous lymphocytes, plasmablasts and pyroninophilic cells, germinal centres were prominent, and the architecture was preserved.

From 0·5 to 0·8% of the total injected radioactivity was concentrated in the popliteal lymph node 2–5 days after injection of ¹²⁵I BEP in FCA. No radioactivity was concentrated in the node after injection of ¹²⁵I BEP without FCA, and animals thus immunized did not develop encephalomyelitis.

The popliteal lymph node was examined by autoradiography after injection of ¹²⁵I BEP in FCA. At 24 hr radioactive encephalitogen associated with droplets of adjuvant was present mainly in the peripheral sinus and at 48 hr encephalitogen–adjuvant droplets were deposited randomly throughout cortex and medulla. These droplets appeared to represent sites where lymphoid cells acquired their capacity for pathogenic reactivity with their target antigen in the central nervous system.

     

The development of lymphocytes with T- or B-membrane determinants in the human foetus

The development of T- or B-membrane determinants on human foetal lymphoid cells was studied by the direct immunofluorescence technique, using a tetramethyl rhodamine isothiocyanate (TRITC) labelled horse antihuman T-cell conjugate (ATC) for the detection of T lymphocytes and a fluorescein isothiocyanate (FITC) labelled goat antihuman Fab conjugate for the demonstration of Ig-bearing B lymphocytes. Human foetal lymphocytes were also tested for spontaneous rosette formation with sheep red blood cells (SRBC).

Cell suspensions of liver, spleen, thymus, bone marrow and blood of twenty-five human foetuses of 5·5–26 weeks of gestational age have been investigated. ATC-positive lymphoid cells were first seen in the liver at 5·5 weeks; E rosette-forming cells (ERFC) and Ig-bearing lymphoid cells were first found at 9 weeks. ERFC were also present in the thymus at 9 weeks. By 12 weeks, fluorescent B and T lymphocytes were found in bone marrow and spleen. ERFC were also found in bone marrow at this age, but not in spleen. At 15 weeks, more than 80% of blood lymphoid cells had T or B determinants.

A difference in the reactivity of lymphoid cells with the ATC and their capacity to form E rosettes was observed. In liver and spleen, the ATC determinant was detectable before the SRBC receptor. In bone marrow, blood and thymus the ATC determinant was found on a higher percentage of lymphoid cells than was the SRBC receptor when those organs were first investigated. During the entire investigated period of gestation, the majority of lymphoid cells in liver and bone marrow did not react with either of the conjugates, nor did they form E rosettes. In all organs investigated, except in the thymus, lymphoid cells were occasionally seen which reacted with both conjugates. By the 16th week of foetal age, more than 90% of lymphoid cells in thymus, spleen and blood had acquired T- or B-membrane determinants.

     

Effect of RES `blockade' on antibody formation: II. Cytokinetics of the secondary haemolysin response and suppressed immunological `memory' in mice treated with carbon particles

Suppression of the primary immune response by treatment of mice with carbon 1 day before initial immunization markedly interfered with development of immunological `memory', since such mice responded to a subsequent challenge injection of RBCs by formation of mainly IgM PFCs and serum antibody. Appearance of IgG PFCs and 2-ME resistant antibody was delayed several days in these carbon treated animals, indicating failure of a typical secondary response. The immune response of these animals was similar to that of a primary response of control animals to a single injection of red cells.

Reticulo-endothelial cell blockade with colloidal carbon suspensions interfered with development of a normal secondary type immune response to sheep red blood cells, as assayed on both the cellular and humoral levels. Fewer antibody PFCs, mainly 19S IgM but also 7S IgG, appeared in spleens of antigen primed mice treated with carbon 1–3 days prior to a challenge injection of red cells, as compared to control primed mice injected with erythrocytes alone. However, the peak day of antibody response was the same for both control and carbon treated animals.

Mice treated with carbon 1–3 days before secondary immunization had much lower peak serum titres, mostly susceptible to 2-ME inactivation.

The time of inoculation of carbon in relation to immunization was important since carbon treatment 1–3 days before secondary RBC immunization resulted in maximum suppression. Injection of carbon 5–7 days before resulted in only a slight effect, whereas injection 30 days before had no detectable effect. Injection of carbon simultaneously or after RBC injection had little effect.

The dose of carbon used for immunosuppression, as well as the concentration of sheep erythrocytes used for immunization affected the number of antibody PFCs and the serum titres in control as well as carbon treated animals.

     

Characterization of human lymphoid cell-mediated antibody-dependent cytotoxicity (LDAC)

Cytotoxicity was studied in a model system using chicken erythrocytes (Ch RBC) labelled with ⁵¹Cr as target cells and human peripheral blood lymphoid cells as effector cells. In vitro human lymphoid cells are highly efficient in destroying target cells coated with anti-target cell antibody, the mean net percentage cytotoxicity of lymphoid cells from fifty-eight control subjects being 64·27±2·06 (SEM). In the absence of antibody the mean net percentage cytotoxicity was 6·90±0·46. As little as 10 ng rabbit anti-Ch RBC IgG was required to cause significant target cell lysis. Studies on the nature of the lymphoid cell-dependent cytotoxic antibody showed that it is localized in the 7S IgG region of whole serum and that an intact Fc region is required; the F(ab')₂ fragment obtained by pepsin digestion of IgG was inactive although able to inhibit the cytotoxic activity of the whole undigested IgG. Investigation of the kinetics of LDAC showed that when antibody was added to the final culture medium target cell lysis progressed rapidly (detectable within 2 hr) and linearly with time up to 8 hr. Thereafter the rate of lysis decreased reaching a maximum after 12 hr culture. With cultures containing target cells which have been pre-incubated with antibody, lysis occurred even more rapidly, detectable within 30 min and reaching a maximum after only 3–4 hr culture. The maximum cytotoxicity in this system was, however, lower than that obtained when antibody was added directly to the culture medium. Cytotoxicity could be inhibited by the addition of aggregated human IgG, as little as 5 μg causing 100% inhibition of target cell lysis. Study of the nature of the effector lymphoid cell showed, first, that viable cells were required, twice frozen/thawed lymphoid cell suspensions being inactive; secondly, that active protein synthesis by the effector cell was not an essential prerequisite, pretreatment of lymphoid cells with mitomycin C having no significan effect on their ability to lyse antibody-coated target cells but significantly reducing their ability to transform in response to the mitogen PHA; thirdly, that the effector cell is non-phagocytic, non-plastic or glass-adherent and does not bear surface immunoglobulin; and fourthly that significant cytotoxicity is detectable even with a total lymphoid cell to target cell ratio of 1:2.     

Kinetics of the proliferative response to antigen in vitro of rabbit lymph node cells taken at various times after immunization

The antigen stimulation of thymidine uptake by immune rabbit lymph node cells in vitro was studied. The kinetics of the response varied depending on the time between immunization and culture. Cultures set up early after immunization showed a peak response over day 1–2 of culture while those set up late after immunization showed a peak response over day 3–4.

Studies using the metabolic inhibitor BUDR suggested that this is due at least in part to a larger recruitment of cells into the response during the third day of culture when lymph node cells taken late after immunization were used.

Removal of antigen from cultures after brief exposure of cells at 4° reduced the magnitude but did not eliminate the proliferative response, suggesting that some antigen is specifically bound to the cells in the cold. Readdition of antigen restored normal reactivity.

Holding cells at 4° for 4 hours without antigen had no effect on their response to subsequent addition of antigen. However, if cells were held at 4° for 3 hours with antigen present a severe degree of depression of subsequent thymidine incorporation was observed in some but not all experiments. This depression of responsiveness was interpreted as an in vitro phenomenon comparable to immunologic tolerance.

     

Correlation of disease activity and Clq-binding immune complexes with the neutrophil inclusions which form in the presence of SLE sera

Intracytoplasmic inclusions containing immunoglobulin (Ig) and complement (C3) are found in normal neutrophils (PMN) after incubation with sera from patients with SLE. These inclusions are believed to be immune complexes removed by phagocytosis from the SLE patients' sera in vitro. Similar inclusions were also noted in the circulating PMN from some patients with SLE. In the present study we have examined the relationship between the presence of intracytoplasmic inclusions and various clinical and laboratory features of SLE. Blood from forty-five patients with SLE was drawn and separated at 37°C. Fresh heparinized blood was also obtained from normal volunteers and allowed to stand for 90 min at 37°C. The buffy coat cells from both normal and SLE groups were removed, centrifuged, washed and examined (direct method) or incubated in the SLE sera for 90 min at 37°C (indirect method). Slides of washed cells were prepared in the cytocentrifuge, stained with fluorescein-conjugated goat anti-human IgG, IgM, IgA and C3 and examined under ultra-violet light.

By the direct method, 24% of patients had small intracytoplasmic inclusions in their neutrophils when stained for IgG suggesting that immune complexes were phagocytosed in vivo. None of twenty-one normal controls had similar inclusions. By the indirect method, 62% of SLE patients were positive for IgG, 15% for IgM, 8% for IgA and 31% for C3. None of the twelve normal controls were positive.

By the indirect method, PMN inclusions containing both IgG and IgM correlated with clinical activity (P<0·001), depressed serum complement (CH50, P=0·026; and C3, P<0·051), cryoglobulinaemia (P=0·014), anti-nDNA antibodies (P<0·001) and Clq-binding immune complexes (P=0·008). A suggestive correlation with granulocytopenia was also observed. The presence of inclusions containing IgG alone did not correlate with any of these parameters. C3 and IgM appeared to be mutually exclusive, i.e. neither was present simultaneously. These findings suggest (1) that normal PMN on exposure to SLE sera develop intracytoplasmic inclusions by phagocytosis of immune complexes, (2) the presence of such complexes correlates with a number of parameters of disease activity, particularly when IgG and IgM are both present and (3) such complexes may be phagocytosed in vivo as suggested by the presence of inclusions in vivo and contribute to a number of granulocyte disturbances seen in patients with SLE. These abnormalities in granulocyte function may be important, predisposing factors for infection in patients with active SLE.

     

Smooth muscle antibody in bronchial asthma

Circulating IgG antibodies reactive with smooth muscle antigens have been found in 21% of patients with intrinsic asthma contrasting with an incidence of 2·9% in extrinsic asthma and 3·7% in chronic bronchitis. This antibody is neither organ nor species specific and does not cross-react with cardiac or skeletal muscle.

In some sera the pattern of fluorescence is distinct from that commonly seen in patients with liver disease and has a `streaky' appearance outlining the surface of individual muscle fibres, similar to the inter-myofibrillary pattern of some cardiac antibodies.

Other autoantibodies were found in seven of the eleven positive sera, suggesting a heightened immunological reactivity in these patients with intrinsic asthma.

There is no evidence from this study that the presence of smooth muscle antibodies is related to pathogenesis of asthma.

     

Circulating complexes in leprosy studied by the platelet aggregation test. The platelet aggregation test and its relation to the rubino test and other sero-immunological parameters in 135 patients with leprosy

Sera from 135 patients with leprosy were tested by the platelet aggregation test (PAT), by the Rubino test and by other sero-immunological assays. PAT positivity (titre≥10) was 53% in the lepromatous subgroups and 5% in the tuberculoid subgroups (P<0·005). The higher PAT titres and Rubino titres clustered significantly (P<0·0005) toward the lepromatous end of the disease spectrum. A statistically significant correlation was found between the PAT and the Rubino titres (0·05>P>0·025). Removal of the effect of the disease spectrum, however, resulted in a partial correlation between the PAT and the Rubino titres that was not significant (P>0·1), suggesting different basic mechanisms for the platelet aggregation (PA) and the Rubino activity of the lepromatous sera. The correlation between the PAT titres and twenty-nine other sero-immunological parameters was calculated, and a highly significant correlation was found between the PAT and the IgG level (P<0·005) and between the PAT and the antistaphylolysin-α titre (P<0·005).

The PA activity in most lepromatous sera studied sedimented in the heavy (>19S) fractions and was inhibitable by IgM rheumatoid factor. It thus fulfilled the criteria for IgG complexes as defined in previous studies with known model Ag/Ab complexes and with sera from patients with immune complex states. The addition of an excess of soluble mycobacterial antigens affected the PA activity of some lepromatous sera, which suggests that the putative complexes were composed of mycobacterial antigens complexed with corresponding IgG antibody.

It was concluded that the PAT is a sensitive detector of IgG complexes peculiar to the lepromatous leprosy. In leprosy the discriminatory power of the PAT seems to be superior to that of other immune complex tests recently applied for the analysis of leprosy series.

     

Thyroid-stimulating antibody (TSAb) detected in sera of Graves'' patients using human thyroid cell cultures

As a homologous system is required to evaluate the effect of thyroid-stimulating antibody (TSAb) present in the serum of Graves' patients, primary cultures obtained from normal human thyroid gland have been used and the stimulatory effect measured as an increase of cAMP intracellular levels.

Monolayer cell cultures were stimulated by IgG purified from sera of Graves' patients or control subjects and compared to the effect of bovine TSH. Bovine TSH produced a dose-dependent increase in cAMP intracellular levels between 0·05 mU and 2·5 mU/ml, reaching a maximal value after 30 min with higher doses. While normal IgG had no effect, IgG prepared from untreated patients with frank Graves' disease elicited a significant increase in cAMP accumulation at a concentration between 0·05 and 0·5 mg/ml within 60 min in thirteen out of fourteen patients. A longer incubation period showed no further increase in cAMP values, even if in one case a higher concentration (5·0 mg/ml) of Graves' IgG had a delayed response. When the cAMP intracellular level modifications produced by Graves' IgG preparations in thyroid cell cultures were compared to those evoked in thyroid slices, an identical percentage (93%) of positive cases was obtained, without a coincidence of negative cases. Using thyroid slices the cAMP intracellular increase above basal levels was higher, if considered as a percentage, but in cultured cells a very low IgG concentration was sufficient to detect the presence of TSAb. No correlation between the two assays was found.

In conclusion, normal human cultured thyroid cells appeared to be a more suitable substrate when compared to human thyroid slices for detecting the presence of TSAb in Graves' disease and for studying its effect on thyroid cells. However, a 100% TSAb positivity was present in our Graves' patient series only when both assays were used.

     

The relationship of antibody-forming cells to rosette-forming cells

Rosette-forming cells (RFC) increased in numbers in the spleens of mice following injection of sheep red blood cells (SRBC). Very sensitive assay system for detecting plaque-forming cells (PFC) showed that RFC and PFC were present in approximately equal numbers at the height of the immune response. Thereafter PFC numbers declined much more rapidly than RFC.

Two techniques were used to study the contribution of PFC to RFCs (a) velocity sedimentation through foetal calf serum gradients and (b) transfer of individual RFC by micromanipulator into the PFC assay gel containing complement and rabbit antimouse IgG antiserum to identify what proportion of RFC were secreting antibody.

It was shown that, when rosettes were prepared at 4°, <10 per cent were formed by PFC at 4 days after immunization, and <2 per cent at 6 days. Rosettes prepared at 37° contained up to 16 per cent PFC.

It was concluded that PFC had either no cell bound antigen-binding receptors or that the receptors were not demonstrable at 4° by the particular rosette preparation used in the study.

Rosettes prepared late in the immune response were more resistant to mechanical agitation than those prepared early in the response.

     

Effect of RES `Blockade' on antibody-formation: I. Suppressed cellular and humoral haemolysin responses in mice injected with carbon particles

Antibody formation to sheep erythrocytes, as detected both at the cellular and humoral level, was suppressed in adult mice after reticulo-endothelial cell (RES) blockade induced by intraperitoneal injection of colloidal carbon. Fewer antibody plaque-forming cells (PFC) appeared in spleens of carbon pretreated mice as compared to normal controls following intraperitoneal immunization with sheep erythrocytes. The day of peak antibody response was the same, however, for both control and carbon treated animals.

There was no compensatory increase in the number of PFCs in other lymphoid organs of carbon treated animals. Similarly, carbon inoculation had no detectable effect on the number of `background' PFCs in the spleens of unstimulated mice.

The time of injection of carbon in relation to time of immunization influenced the effect since injection of carbon 24–48 hours prior to RBC injection resulted in maximum immunosuppression. Injection of carbon 4–5 days before red blood cells resulted in only partial immunosuppression. Treatment with carbon 1–2 weeks prior to immunization had no detectable effect. Similarly, injection of carbon 1 or 2 days after immunization had little or no effect on the peak plaque response.

The decrease in the amount of serum antibody to sheep red blood cells in carbon treated mice was not as marked as that which occurred on the individual cell level. However, most of the antibody in the sera of carbon treated animals was susceptible to 2-mercaptoethanol, even 1 or 2 weeks after immunization. On the other hand, serum antibody from control mice was mainly 2-mercaptoethanol sensitive only during the first week after immunization.

Immunosuppression seemed to be related to a direct effect of carbon since the supernatant fluid obtained from carbon suspensions was not suppressive. Also, washed or dialysed carbon preparations were just as effective as the original preparation in suppressing antibody responses.

     

Quantitative determination of hepatitis B antigen by means of radial immunodiffusion

The method of radial immunodiffusion for measurement of hepatitis B antigen (HB ag) levels in sera of twenty and urines of three HB ag positive patients is described. In patients suffering from acute hepatitis, the level of HB ag in serum was 0·15–2·12 mg/ml (mean = 1·08±SD 0·69 mg/ml). In patients suffering from chronic hepatitis the level of HB ag in serum was 0·36–3·61 mg/ml (mean 2·56±SD 1·13 mg/ml). The highest levels of HB ag in serum, 2·62–5·12 mg/ml (mean 3·91±SD 0·90 mg/ml) were observed in symptomless HB ag carriers.

The amounts of HB ag excreted in urine, found in two chronic hepatitis patients and one HB ag carrier, were 13·2 mg, 46 mg and 82 mg/1000 ml of urine respectively.

     

In vitro stimulation of antibody formation by peritoneal cells: III. Effect of active immunization on the subsequent in vitro performance of peritoneal and spleen cells

Male CBA mice were given a single intraperitoneal injection of sheep red blood cells (SRBC) or horse red blood cells (HRBC). They were killed at intervals of 1–10 days thereafter, and micro-cultures of spleen cells or peritoneal cells (PC) were prepared. These consisted of a thin film of tissue culture medium containing carboxymethyl cellulose (CMC), mouse lymphoid cells, guinea-pig complement and either SRBC or HRBC, held at 37° under liquid paraffin. Cultures were read repeatedly for appearance of haemolytic plaques.

PC from SRBC-immunized mice showed an altered reactivity on SRBC monolayer cultures. The peak plaque count achieved in vitro fell progressively for 4 days after immunization, and then returned to normal by day 7. The actinomycin D resistant component of the PC response rose rapidly; at 1 day after immunization it was equal to the total response. Over the next 3 days after immunization it fell again to normal levels. The results suggested that the in vivo injection sets in train events locally in the peritoneal cavity which resembled those following in vitro culture of normal PC in SRBC monolayers. The effects were immunologically specific as only marginal changes followed the injection of HRBC.

Spleen cells from SRBC-immunized mice, when cultured in SRBC monolayers, yielded many cells capable of giving plaques after 5–60 minutes incubation, as expected. These were deemed to be cells forming antibody at the moment of killing of the animal. In addition, such cultures developed new plaques over the subsequent 23 hours in culture. These were produced by cells not initially forming antibody which switched into antibody secretion at some time during culture. At early time points after immunization, this second type of cell was much more numerous than the first type. The switch from non-secretor status could occur in the presence of a high concentration of actinomycin D. Operationally these non-secretors in immunized spleens resembled an important fraction of PC from unimmunized retired breeder mice. The progressive conversion of non-secretor cells into secretors, if it occurs in vivo, would have a major influence on the kinetics of appearance of PFC in a spleen after immunization.

While spleen cells from mice immunized with HRBC performed on HRBC monolayers much as described above, PC from HRBC-immunized mice could not be induced to cause significant lysis in HRBC monolayers. The same was true of PC from mice chronically fed with HRBC. In fact, no method has yet been found to persuade PC to produce lytic plaques active against erythrocytes other than SRBC.

     

Suppression of primary immunization to the rabbit red cell alloantigen HgA by passively administered anti-HgA

Hg^A-negative rabbits were given an intravenous injection of Hg^A-positive red cells followed within 30 minutes by an intravenous injection of IgG anti-Hg^A. Several weeks later a second injection of Hg^A-positive red cells, without antibody, was given; the survival of these red cells was compared with the survival of red cells given to animals which had received no passive antibody. As judged by a relatively good survival of the second injection of red cells, primary immunization to Hg^A was at least partially suppressed when the dose of anti-Hg^A given with the first injection of red cells was such that 260–750 μg antibody was bound per m1 red cells. In three groups of animals given Hg^A-positive cells and anti-Hg^A in doses such that 20–40 μg antibody was bound per m1 red cells there was apparently no suppression of the antibody response.

It is concluded that the amount of specific IgG antibody required for the suppression of primary immunization is at least ten times greater, in terms of μg antibody per m1 red cells, for the rabbit antigen Hg^A than for the human antigen Rh; the difference may be due partly to the greater number of Hg^A antigen sites on the rabbit red cell compared with the number of Rh(D) sites on the human red cell.

     

Serum associated leucocyte locomotion inhibition and leucocyte motility in malignant melanoma: effect of BCG treatment

Serum-associated leucocyte locomotion inhibition (SALLI) and leucocyte motility were investigated in patients with malignant melanoma. Ten days after tumour excision twelve out of eighteen patients' sera exhibited a SALLI exceeding the normal range of 15%. The mean SALLI thus reached was 59·2±5·2%. No correlation was observed between SALLI and the level of invasion or the stage of the disease.

Six patients selected at random who had a mean SALLI of 71·5±5·8% after tumour excision were further treated by BCG immunotherapy and presented after 8·2±2·9 months of therapy with a significantly (P<0·01) lower SALLI of 32·6±8·1%. In eight patients treated exclusively by surgical excision, SALLI remained basically unchanged in the course of 10±2·8 months (29·0±8·0% vs 30·4±12·9%).

The mean index of leucocyte locomotion (LL) of eight melanoma patients who had received BCG for 11·2±2·3 months was 5·9±0·9 cells/field and thus significantly (P<0·01) higher in comparison with the mean index of LL (2·8±0·5) found in eight patients treated by surgical excision only 12·4±2·1 months before testing.

In addition, patients receiving BCG had a significantly higher (P<0·05) mean value of LL than fifteen healthy controls who presented with a mean index of LL of 3·4±0·3 cells/field. Our results permit the suggestion that BCG decreases SALLI and increases LL in melanoma patients.

     

In vitro preferential effect of irradiation on cultured T lymphoid cell line

The present report describes a comparative study of cytocidal effect of irradiation on human cultured T and B lymphoid cells. Cytocidal effect of irradiation was expressed as percentage inhibition or percentage reduction, which was calculated by comparing the [³H]thymidine incorporations or viable cell counts in irradiated and unirradiated cultures, respectively. A higher percentage inhibition was observed in each and every T-cell culture, irradiated with 100–4000 rads at days 1–4 of incubation. The most significant difference of percentage inhibition between T and B cells (P<0·05–P<0·01) was observed at each irradiation dose level at day 3. A significant percentage reduction of viable T cells (98–100%) was noted at 100 rad dose level, whereas only 0–3% of B lymphoid cells were killed by this dose of irradiation at days 2–4 of incubation. Our data clearly indicate that human cultured T lymphoid cells are extremely radiosensitive and B lymphoid cells, on the other hand, are fairly radioresistant.     

Laboratory evaluation of anti-phospholipid syndrome: a preliminary prospective study of phosphatidylserine/prothrombin antibodies in an at-risk patient cohort

Immunoglobulin (Ig)G/IgM autoantibodies to phosphatidylserine/prothrombin (aPS/PT) were evaluated individually and in combination with criteria anti-phospholipid (aPL) tests in a prospectively ascertained cohort of patients at risk for anti-phospholipid syndrome (APS). One hundred and sixty (160) consecutive requests for lupus anti-coagulant (LAC) from the University of Utah Health Sciences Center were identified during 8 weeks. Of these, 104 unique patients had additional requests for cardiolipin (aCL) and/or beta2 glycoprotein I (aβ₂GPI) IgG and/or IgM; samples were retained and analysed for aPS/PT, aCL and/or aβ₂GPI IgG and IgM antibodies. Following testing, a comprehensive chart review was performed and patients categorized according to their clinical diagnosis. Individual and combined sensitivities, specificities, odd ratios (OR), diagnostic accuracy for specific tests or combinations by receiver operating characteristic (ROC), area under the curve (AUC) analyses and correlations between test results were determined. The sensitivities of aPS/PT IgG/IgM (54·6/45·5%) were lower than LAC (81·8%) but higher relative to aCL IgG/IgM (27·3/0%) or aβ₂GPI IgG/IgM (27·3/0%). The best correlation between LAC and any aPL test was observed with aPS/PT (P = 0·002). There was no significant difference in the diagnostic accuracies for any panel with LAC: LAC/aβ₂GPI IgG/aCL IgG [AUC 0·979, OR 475·4, 95% confidence interval (CI) 23·1–9056·5, P = 0·0001 and LAC/aβ₂GPI IgG/aPS/PT IgG or LAC/aPS/PT IgG/aCL IgG (AUC 0·962, OR 265·3, 14·2–4958·2, P = 0·0001). The high correlation between LAC and aPS/PT IgG/IgM in this preliminary study suggest that this marker may be useful in the evaluation of APS. More studies to determine the optimal aPL antibody tests combination are needed.