Gamma globulin complexes in synovial fluids of patients with rheumatoid arthritis. Partial characterization and relationship to lowered complement levels

Gamma globulin complexes were demonstrable in certain joint fluids from patients with rheumatoid arthritis by analytic and density gradient centrifugation. They form a continuum of high molecular weight components ranging from 7S to 30S and were dissociable primarily to 7S γG globulin. The larger complexes were also detectable by precipitation reactions with C1q and with γM rheumatoid factor. This permitted the isolation and partial characterization of the complexes. Non-immunoglobulin constituents were not detectable. Evidence was obtained that 7S γG globulin rheumatoid factors represented an important constituent of the complexes.

A relationship was encountered between the amount of γ globulin complex present in the joint fluids and diminution in total haemolytic complement activity. All fluids with abundant γ globulin complexes contained markedly lowered complement levels. A decrease in levels of C1q and β_1A was found to correlate with the amount of γG globulin complexes. Although patients who had diminished complement levels in their joint fluid have serum γM rheumatoid factor, its titre does not correlate well with the extent of complement depression.

Joint fluids with abundant γ globulin complexes manifested an anticomplementary effect. This activity was most apparent at 37°C when fresh rheumatoid serum was used as a source of complement. Evidence indicating the participation of γM rheumatoid factor in this anticomplementary effect was obtained. All fluids with significant anticomplementary activity formed precipitin bands with C1q on agarose plates. γM rheumatoid factor was not necessary for this reaction.

     

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.

     

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.     

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.

     

Hepatitis C virus (HCV)-induced IgG-IgM rheumatoid factor (RF) complex may be the main causal factor for cold-dependent activation of complement in patients with rheumatic disease

A low serum complement level is commonly found in patients with rheumatic diseases. We evaluated 170 patients with such diseases to determine their serum levels of CH₅₀, C3 and C4 protein. Persistent hypocomplementaemia was found in 19 of those patients, particularly in those with systemic lupus erythematosus (SLE). Cold-dependent activation of complement (CDAC) was demonstrated in nine of the 19 (47.4%), and six of the nine patients demonstrated infection with HCV (66.7%). The nine patients that exhibited CDAC had nearly normal haemolytic complement activity when the sera were separated either at 37°C or in EDTA-treated plasma. Conversely, it markedly decreased, even to the point of being immeasurable, when the sera were separated at 4–21°C. No significant deficiency in C3 and C4 protein levels was found in these patients. Clinical parameters other than levels of anti-HCV antibody, transaminase, and RF were not influenced by CDAC. In an attempt to isolate the causal factor for CDAC, we isolated IgG fractions from the CDAC patients by using a protein G column, in which case precipitates were collected from the eluates. The precipitates were mixed with normal serum and incubated at 4–21°C for 18 h. A decrease in the level of CH₅₀ in normal serum was observed, which predominated (P<0.001) when precipitates from HCV-infected patients were used. This indicated CDAC was possibly interrelated to the precipitates of such patients. This precipitate was proved to contain IgM besides IgG. It is therefore possible that an HCV-related IgG complex or an IgG-IgM RF complex may be formed at low temperature and be involved in activating the complement system in vitro.     

The Behaviour of human vitronectin in vivo: effects of complement activation, conformation and phosphorylation

We examined the behaviour in vivo of native, specifically phosphorylated, and multimeric vitronectin to determine the effects of these modifications on its turnover, distribution and molecular behaviour. In normal rabbits, the plasma half-life (T_1/2) of antigenically detected vitronectin was 8.00 ± 1.26 h (mean ± s.d.), with a fractional catabolic rate (FCR) of 18.77 ± 1.57%/h and extravascular/intravascular ratio (EV/IV) of 1.00 (0.48–1.60, median and range). For vitronectin selectively phosphorylated by protein kinase A, T_1/2 was 8.87 ± 0.48 h, with a significantly smaller FCR of 10.85 ± 0.71%/h (P<0.005) and an EV/IV of 0.28 (0.15–0.36) (P<0.05 compared with antigenically detected vitronectin). In vitro, phosphorylation had no effect on the affinity of vitronectin for heparin–Sepharose, while complement activation with cobra venom factor (CVF) led to a two-fold enrichment of ³²P-vitronectin within the SC5b-9 complex. In vivo CVF caused a rapid decrease in the circulating levels of ³²P-vitronectin and was accompanied by the prompt appearance of a high mol. wt species consistent with SC5b-9. Despite specific incorporation of ³²P-vitronectin into SC5b-9, both forms of the molecule had similar inhibitory effects on C9-mediated haemolysis of EAC1-7 cells. Urea-activated vitronectin was rapidly cleared from circulation with less than 15% remaining after 1 h while protein-bound label accumulated in the spleen, lung and liver. These results demonstrate that vitronectin is a rapidly metabolized protein whose in vivo behaviour is markedly altered when phosphorylated or activated to form multimers and SC5b-9.     

Metabolism of radio-iodinated IgG in patients with abnormal serum IgG levels. II. Hypogamma-globulinaemia

The metabolism of radio-iodinated IgG was studied in seven patients with IgG deficiency (<600 mg/100 ml). The group comprised two patients with primary hypogamma-globulinaemia, one with primary hypogamma-globulinaemia and nodular lymphoid hyperplasia, and one each with chronic lymphatic leukaemia (CLL), γG multiple myeloma, renal homotransplantation and the nephrotic syndrome.

All patients had markedly reduced plasma and total body pools of normal IgG with normal distribution (intravascular and extravascular pools). An increase in the initial urinary excretion of free isotope in one patient was attributed to a degree of protein denaturation. Compared to the normal range of 20–60 mg/kg/day for IgG synthesis, the patients with primary deficiency and the patient with CLL all had markedly reduced synthesis of normal IgG (2, 5, 4 and 7 mg/kg/day respectively). These patients also had a prolonged or normal plasma T½ and a normal or decreased fractional turnover rate (FTR).

The patients with deficiency of normal IgG secondary to multiple myeloma, renal homotransplant or nephrotic syndrome all had an IgG synthesis rate at the lower limit of the normal range with values of 19, 21 and 19 mg/kg/day respectively. They had increased catabolism with a short plasma T½ and increased FTR.

An assessment of the factors controlling the metabolism of normal IgG is important in the individual patient as regards the management of their recurrent infections with therapeutic human γ-globulin.

     

The catabolism of human γG immunoglobulins of different heavy chain subclasses. III. The catabolism of heavy chain disease proteins and of Fc fragments of myeloma proteins

The catabolism of ¹³¹I and ¹²⁵I paired labelled Fc fragments of myeloma proteins and of H chain disease proteins of different heavy chain subclasses was studied in men and monkeys. In contrast to the previously demonstrated catabolic heterogeneity of intact γG immunoglobulins, the Fc fragments and H chain disease proteins of all subclasses tested were catabolized at a similar rate. These data suggest that structures not present on the Fc fragments are responsible for the faster turnover rate of γG₃ immunoglobulins and for the differences in half-lives of myeloma proteins within a given subclass.The catabolic features of the H chain disease proteins differed from those of intact γG. Although the whole body half-lives of the two proteins were similar, the fractional turnover rate of the H chain disease proteins was higher than that of γG, on the average 8% of the intravascular pool/day as compared to 4% for γG. One-half to 1% of the intravascular pool of the H chain disease protein and less than 0·1% of the γG was excreted into the urine. An average of 24% of the H chain disease proteins and 44% of the γG equilibrated into the intravascular compartment.     

Blood monocyte activation in rheumatoid arthritis: increased monocyte adhesiveness, integrin expression, and cytokine release

Infiltration of the synovium by mononuclear cells, namely lymphocytes and monocytes, is one of the main features of rheumatoid arthritis (RA) and is considered to be responsible for the development of the disease. In this study in 31 consecutive patients with RA, we investigated whether peripheral blood monocytes exhibited markers of cellular activation related to cell migration. Using flow cytometry with the respective specific antibodies, we studied the expression of integrins CD11a, CD11b, CD11c, CD49d (VLA-4), and CD49e (VLA-5) on monocytes from patients with RA and from normal (N) subjects. IL-1β, IL-6, and tumour necrosis factor-alpha (TNF-α) production by cultured monocytes was measured by immunoassay. Adhesiveness of monocytes was studied on various surfaces (plastic, human fibronectin, gelatin-coated plasma, subendothelial matrix) and on cultured endothelial cells under basal conditions or after stimulation by IL-1β. An increased number of CD14⁺ monocytes (Mo) from RA patients expressed the CD11b molecule (RA Mo = 90·3%, N Mo = 83·4%, P < 0·005). The expression of CD11b on CD14⁺ monocytes was significantly increased in RA patients (median fluorescence intensity (FI): RA Mo = 145 (range 80–466) units; normal Mo = 95 (range 24–164) units; P < 0·003). Production of extracellular IL-1β and IL-6 by RA monocytes was significantly enhanced compared with monocytes from normal subjects (IL-1β: RA = 2·65 ± 0·91 ng/ml versusN = 1·35 ± 0·85 pg/ml, P < 0·05; IL-6: RA = 4·83 ± 0·90 ng/ml versusN = 2·40 ± 0·95 ng/ml, P < 0·05). Compared with normal monocytes, RA monocytes exhibited increased adhesion to the various surfaces studied (plastic, P < 0·01; fibronectin, P < 0·01; and gelatin-coated normal or RA plasma, P < 0·01) as well as to unstimulated (P < 0·01) and IL-1β-stimulated endothelial cells (IL-1β for 4 h, P < 0·05; IL-1β for 24 h, P < 0·05). In our study, blood monocytes from RA patients exhibited features of activation related to cell adhesion.     

The effect of diameter on the electrical constants of frog skeletal muscle fibres

1. Electrical constants were determined on isolated single fibres or on fibres from bundles from frog's twitch muscles by analysing the low frequency cable properties.

2. The sarcoplasmic conductivity (G_i) was 5·9 mmho/cm at 20° C, and its temperature coefficient (Q₁₀) was 1·37.

3. The Q₁₀ of the membrane conductance (G_M) was 1·49, and that of the membrane capacity (C_M) was 1·02.

4. C_M increases with diameter (D) in an approximately linear manner: the values were 4·6 μF/cm² at D = 50 μ, and 8·5 μF/cm² at D = 130 μ.

5. G_M also increases with diameter, being 0·21 mmho/cm² at D = 50 μ and 0·37 mmho/cm² at D = 130 μ.

6. These results suggest that the transverse tubular system contributes substantially to the values of low frequency capacity and conductance measured at the surface membrane.

     

Increased numbers of CD5+ B cells and T cell receptor (TCR) γδ+ T cells are associated with younger age of onset in rheumatoid arthritis (RA)

Patients presenting with RA before the age of 45 years (younger onset) are known to have more aggressive disease compared with patients presenting after the age of 65 years (older onset). Coordinated expansion of circulating CD5⁺ B cell and TCR γδ⁺ T cell levels has been reported in patients with RA. This study assesses the peripheral blood levels of these two cell types in RA patients with younger and older onset of disease. CD5⁺ B cell levels were significantly elevated in the younger onset RA group (26·6 ± 4·5%) compared with the older onset RA group (14·2 ± 1·2%; P <0·01). TCR γδ⁺ T cell levels were also significantly raised in the young patients (4·0 ± 0·9%) compared with elderly patients (1·6 ± 0·2%; P <0·01). T cell levels (CD3⁺) were similar in both groups (young 66·4 ± 3·3%; old 74·3 ± 3·4% (mean ± s.e.m.); NS). Total B cell levels (CD19⁺) were also similar in these groups (7·7 ± 0·7% versus 8·9 ± 1·8%; NS). A significant positive correlation was observed between the CD5⁻ B and TCR γδ⁺ T cell types in the patients (r = 0·72, P <0.05). Compared with age-matched normal controls, the younger onset patients had similar CD5⁺ B cell and TCR γδ⁺ T cell levels to the elderly controls (CD5⁺ B cells 30·2 ± 3·0%; TCR γδ⁺ T cells 3·0 ± 0·8%). Conversely, older onset RA patients had CD5⁺ B cell levels similar to the young controls (12·3 ± 1·9%). Spontaneous in vitro synthesis of immunoglobulins (IgM, IgA and IgG) and rheumatoid factors (IgM and IgA isotypes) were not significantly different in both patient groups. The coordinate expansion of circulating CD5⁺ B cells and γδ⁺ T cells seen in patients with RA presenting before 45 years of age and not after 65 years of age may suggest a potential role for these cells in more aggressive disease states.     

Distribution of chylomicrons and albumin in dog kidney

1. Under specified experimental conditions the distribution space of labelled chylomicrons in the kidney was 13·8 ± 0·9 ml./100 g. tissue. The assumption is supported that this provides a measure for the quantity of intravascular plasma constituents.

2. Values for red blood cells and albumin distribution spaces were 5·2 ± 0·6 and 20·2 ± 1·0 ml./100 g tissue, respectively, in the whole kidney. The ratio of tissue haematocrit over simultaneous arterial haematocrit averaged 0·56. The extravascular albumin fraction amounted to about 31·0% of the total albumin in the whole kidney.

3. A statistically significant correlation was demonstrated between osmotic urine/plasma (U/P) ratios (within the approximate limits of 0·6-1·8) and quantities of extravascular albumin in the medulla.

     

Calreticulin promotes angiogenesis via activating nitric oxide signalling pathway in rheumatoid arthritis

Calreticulin (CRT) is a multi-functional endoplasmic reticulum protein implicated in the pathogenesis of rheumatoid arthritis (RA). The present study was undertaken to determine whether CRT was involved in angiogenesis via the activating nitric oxide (NO) signalling pathway. We explored the profile of CRT expression in RA (including serum, synovial fluid and synovial tissue). In order to investigate the role of CRT on angiogenesis, human umbilical vein endothelial cells (HUVECs) were isolated and cultured in this study for in-vitro experiments. Our results showed a significantly higher concentration of CRT in serum (5·4 ± 2·2 ng/ml) of RA patients compared to that of osteoarthritis (OA, 3·6 ± 0·9 ng/ml, P < 0·05) and healthy controls (HC, 3·7 ± 0·6 ng/ml, P < 0·05); and significantly higher CRT in synovial fluid (5·8 ± 1·2 ng/ml) of RA versus OA (3·7 ± 0·3 ng/ml, P < 0·05). High levels of CRT are expressed in synovial membrane localized predominantly to inflammatory cells and synovial perivascular areas in both the lining and sublining layers of RA synovial tissue (RAST). Increased nitric oxide (NO) production and phosphorylation level of endothelial nitric oxide synthase (eNOS) were measured in HUVECs following CRT stimulation, while the total eNOS expression was not significantly changed. Furthermore, CRT promoted the proliferation, migration and tube formation of HUVECs, which were significantly inhibited by a specific eNOS inhibitor. These findings suggested that CRT may be involved in angiogenesis events in RA through NO signalling pathways, which may provide a potential therapeutic target in the treatment of RA.     

Experimental induction of rheumatoid factor-like substances in animal trypanosomiasis

Rheumatoid factor-like substances were induced in rabbits by infection with Trypanosoma equiperdum. There was a certain parallelism with the phenomena described earlier with T. gambiense infections in man. The anti-IgG globulins were IgM with a preference for heterologous (human) IgG in the latex fixation test. A correlation was found between the latex fixation titres and the IgM levels in the sera. A naturally occurring pre-infectious agglutinator was not of IgM nature. The anti-IgG globulins developed in all the infected animals, mostly within 2 weeks and often before the IgG levels in the sera started to increase.

The failure to induce rheumatoid factor-like substances in mice infected with a certain strain of T. gambiense indicates the importance of the host–parasite relationship for the formation of rheumatoid factors.

The single radial diffusion method according to Mancini, Carbonara & Heremans (1965) did not give valid results for the determination of IgM in the rabbit sera, but could be used for IgG. IgM was determined by an indirect method.

These experiments might form the basis of a model for investigating the nature of rheumatoid factor formation.

     

The functional activities of IgG and IgM anti-A and anti-B

The rate constants for association and dissociation, and the equilibrium constants, were determined for ¹²⁵I-labelled anti-A and anti-B of both IgG and IgM molecular types. The following results and conclusions were obtained:

1. The equilibrium constants were within the range 06×10⁸–13.0×10⁸ l/mole, and were of the same order for both IgG and IgM antibodies.

2. The initial rate constants for association were in the range 2.1×10⁵–4.8×10⁵ l/mole/sec, and the energy of activation (E_a) 6700–9000 cal/mole. These results indicate that the rate of association is approaching the limit set by the rate of diffusion of the reactants.

3. The initial rate constants for dissociation were 1 × 10^-4–5 × 10^-4/sec and E_a = 20,000–36,000 cal/mole. These latter values suggest that more than one bond must be broken simultaneously during dissociation.

4. Ionic strength and pH changes have only a minor effect on the constants; this indicates absence of ionic groups on A and B antigen sites.

5. The changes in enthalpy were –5400 to –21,800 cal/mole; the reactions are mainly enthalpy driven and this accounts for the fact that anti-A and anti-B agglutination titres increase as the temperature is decresed.

6. There was heterogeneity of the values of the standard change in free energy, enthalpy and entropy within each example of antibody.

7. The approximate concentrations of antibody at the end-points of the agglutination titres were: for IgG antibody, 0.2 μg/ml; for IgM antibody, 0.01 μg/ml.

     

Role of lymphocyte activation products (LAP) in cell-mediated immunity. I. Preparation and partial purification of guinea-pig LAP

Partial purification of soluble products of guinea-pig lymphocyte activation (LAP) was undertaken by fractional precipitation with ammonium sulphate, ion-exchange and Sephadex chromatography, and by immune precipitation of inducing antigen and of contaminating serum protein. During these purification steps the activity of macrophage migration-inhibition factor (MIF) was concentrated up to 1300-fold and separated from inducing antigen and serum protein. An endpoint assay was devised for expressing antigen-induced MIF activity of LAP fractions as weights of material giving 30% inhibition of migration (MI₃₀ doses).

MIF activity precipitated between 50% and 80% saturated ammonium sulphate and eluted from DEAE-cellulose at pH 7·9 at intermediate salt concentrations (0·03–0·2 M phosphate). On Sephadex gel filtration MIF activity was concentrated in fractions of molecular weight range 56,000–82,000 with a smaller amount of activity eluting from 20,000–56,000. After immune precipitation of extraneous protein and elution from DEAE-cellulose, LAP material was found to have an MI₃₀ dose of 0·4 μg.

Materials representative of antigen and serum protein-depleted MIF were selected for intralymphatic injection in order to determine whether MIF-rich LAP fractions were able to induce paracortical distension in guinea-pig lymph nodes (see following paper).

     

The glutamic acid decarboxylase 65 immunoglobulin G subclass profile differs between adult-onset type 1 diabetes and latent autoimmune diabetes in adults (LADA) up to 3 years after clinical onset

Autoantibodies against glutamic acid decarboxylase 65 (GADA) are found frequently in patients with autoimmune diabetes. Immunoglobulin (Ig)G₁ is the most frequent subclass among the GADA IgG subclasses. IgG₄ is a more common subclass in latent autoimmune diabetes in adults (LADA) at clinical onset compared to type 1 diabetes. The aim of this work was to study the different GADA-IgG subclass profiles during a 3-year follow-up in these groups of autoimmune diabetes. Adult-onset subjects, classified as either type 1 (n = 40) or LADA (n = 43), were included in the study. New samples were collected every year from these patients. In addition to conventional GADA analyses, GADA-IgG subclasses were also analysed with a radioimmunoprecipitation assay using biotin-conjugated antibodies (directed against human IgG subclasses and IgM) and streptavidin Sepharose. During 3 years'' follow-up, all the IgG subclass levels decreased in type 1 diabetes – IgG₁: P < 0·001; IgG₂: P < 0·001; IgG₃: P < 0·001; IgG₄: P < 0·05 (Friedman''s’ test) – while levels remained stable for all four subclasses in LADA. GADA IgM, however, decreased in both groups (P < 0·001). Patients with LADA have higher GADA IgG₃ and IgG₄ at clinical onset and seem to maintain the levels and profile of their IgG subclasses up to 3 years after clinical onset, while all the GADA IgG subclass levels decrease in type 1 diabetic patients. This indicates a persistent different immune response in LADA compared to type 1 diabetes and further indicates the difference in pathogenesis.     

The transmissions of antibodies across the gut of pouch-young marsupials

The transmission of antibodies across the gut of suckling pouch-young was investigated in three species of marsupials (Setonix brachyurus, Macropus eugenii and Trichosurus vulpecula) from Australia.

Mother Setonix, immunized against Salmonella adelaide flagella and Bacteriophage Φ × 174, transmitted the antibodies in milk to their young. In sucrose density gradient runs, the antibody activity in milk whey and in serum of pouch-young, of Setonix and Macropus was found to be in the 7S region only; antibody in the 11S and 19S regions was not detected. Chromatographic preparations of IgM antibodies were fed to pouch-young Setonix which were later bled and their serum titrated for anti-S. adelaide agglutinins and antiphage Φ × 174 activity. The IgM antibodies were not transmitted across the gut in detectable amounts.

Antibodies were present in the blood of pouch-young Setonix within 15–60 minutes of gavage (feeding by stomach tube) of immune serum. In Setonix the capacity to absorb antibodies in the intestine was lost at an age between 170 and 200 days and in Trichosurus it was lost at an age between 98 and 145 days. At these ages the pouch-young were able to leave the marsupium for varying lengths of time. Antibodies did not traverse the rumen wall in a young Setonix whose rumen was isolated from the intestine with ligatures before immune serum was gavaged.