Complement and complement-like activity in lower vertebrates and invertebrates.

A purified cobra venom factor with C-inhibiting activity also promotes lysis of erythrocytes in fresh mammalian serum. Lysis-inducing activity of purified cobra venom factor was found in sera of lower vertebrates including the cyclostome hagfish and in invertebrates. Lysis-inducing activity was most effective with frog serum. Frog serum was found to be more hemolytic for E(s) in the presence of CVF than when cells were sensitized with hemolysin. The hemolysis induced by CVF with frog serum, as in the higher vertebrates, was inhibited when sera were pretreated with known C inhibitors including heat, chelators, endotoxin, immune complexes, and CVF itself. Complexes formed with CVF and either frog serum or invertebrate hemolymph promoted lysis of indicator cells in the presence of frog serum in EDTA. This lysis was most marked when the starfish-CVF complex was used and was C-dependent. Conversely, complex formed with frog serum and CVF promoted lysis of E in the presence of invertebrate hemolymph (Limulus) in EDTA. Hence, serum components were to some degree at least interchangeable between vertebrate sera and invertebrate hemolymph. Lysis-inducing activity of purified CVF occurs in a wide range of species, has revealed activities resembling those of terminal C-components in lower vertebrates and invertebrates, and provides one means for study of C and C-like activities in primitive species.

Hereditary C2 deficiency: Genetic studies and association with the HL-A system.

Herediatary C2-deficiency has been shown to be transmitted asn an autosomal recessive characteristic. Recent evidence indicates that some genetic factors involved in the control of the complement (C) system in both man and mice are governed by genes localized within the major histocompatibility regionmthis study describes a large pedigree of the paternal family of a C2-deficient patient with systemic lupus erythematosusl It is shown that this condition is transmitted as an autosomal recessive trait, the heterozygous carriers having approximately half normal levels of C2. Furthermore, this trait was shown to be inherited in close linkage with an infrequent HL-A typw, 2,4A2. The maternal, C2-defective chromosome was shown to be transmitted by HL-AW10, W18 haplotypemthis same haplotype was described in a similar study by Fu et al. (6) to be associated with C2 deficiencymfinally, a third haplotype HL-A2,W18 carrying a defective C2 gene was demonstrated in a part of this pedigree.

Antibody-independent complement activation by myelin via the classical complement pathway.

Murine or rabbit whole brain homogenates were shown to activate human complement via the classical pathway by an antibody-independent reaction. This activity required Ca++ ions. Anticomplementary activity in fractionated murine brain was found to reside in the myelin fraction and in purified myelin. It was absent, however, both from highly purified myelin basic protein (MBP) and from the MBP-free residue. Because purified MBP is a monomer and this protein exists in brain tissue largely as a dimer, the ability of the cross-linked form of MBP to activate complement was investigated. MBP, dimerized with difluorodinitrobenzene, was highly anticomplementary. The murine brain, inactive when taken from the newborn mouse, was shown to first acquire the capacity to activate complement at 7 d after birth. This finding is consistent with the report that the synthesis of myelin protein has been shown to be initiated in murine brain 8 d after birth. Complement activation by MBP could play an important role in the pathological changes observed in neurological disorders.

Mixed lymphocyte culture determinants and C2 deficiency: LD-7a associated with C2 deficiency in four families.

Four families with C2 deficiency were studied. Among eight HL-A haplotypes involved with C2 deficiency, five were HL-A 10,W18. Three homozygotes for C2 deficiency from different families were mutually nonreactive in mixed lymphocyte cultures (MLC) and the heterozygotes from the fourth family failed to react to the homozygous cells. It appeared that identical MLC determinants were associated with all the genes from the different families that related to C2 deficiency. Further experiments identified the MLC determinant, LD-7a, as being involved. These results suggest marked linkage disequilibrium between the genes for C2 deficiency and the major histocompatibility complex (MHC). Studies of possible recombinants have offered tentative evidence for the positioning of the locus for C2 deficiency with respect to other segments of the MHC.

Suppression of gamma interferon production by inactivated feline leukemia virus.

Supernatants from cultures of normal feline lymphocytes stimulated with Staphylococcus enterotoxin A showed antiviral activity, characterized as a gamma-like interferon. With the addition of inactivated feline leukemia virus, markedly less interferon was produced. The reduction in interferon production was not attributable to lowered lymphocyte viability or reduced mitogenic properties of Staphylococcus enterotoxin A and appears to be a direct retroviral effect. This finding may reflect clinically relevant events that may contribute to the development of the feline or human states of acquired immunodeficiency.

Circulating immune complexes detected by 125I-Clq deviation test in sera of cancer patients.

The presence of circulating immune complexes in freshly drawn sera of patients with various forms of malignancies was detected by the 125I-Clq deviation test of Sobel et al. More than 50% of the 459 cancer sera showed a high inhibition of 125I-Clq uptake by sensitized sheep erythrocytes when compared with sera of 50 healthy laboratory personnel. The levels were compared with levels of total hemolytic complement and immunochemical determinations of Cl1 and C3. A correlation between high levels of circulating immune complexes and low levels of Clq was suggested. These immune complexes were separated by sucrose density gradient ultracentrifugation at low pH and were found to be heavier than 19S. Fluctuation of levels of immune complexes was evident when serial samples from the same patient were tested. Decrease of levels of immune complexes and a concomitant increase of Clq were detected after Calmette-Gueérin bacillus and autologous tumor cell treatment in some melanoma patients.

Circulating immune complexes in sera of children with neuroblastoma: correlation with stage of disease.

The presence of circulating immune complexes (ICS) in freshly drawn sera of 67 children with neuroblastoma was studied by the Raji cell radioimmunoassay of Theofilopoulos et al. (J. Clin. Invest. 57: 169--182), with particular emphasis on the correlation of levels of ICS with stage of disease and changes attributable to treatment. There was a close correlation between amount of complexes and stage of disease and treatment. Levels of ICS increased as the stage of the disease advanced, and were significantly higher (P less than 0.005) in stage IV than in all other stages combined. When patients with stage IV disease were subdivided into "before," "during," and "after" treatment groups, there was a significant decrease in ICS levels as treatment progressed. Studies of complement and complement components did not give such a clear relationship. A significant decrease of hemolytic C1 values was found in patients with "active disease" compared to normal age-matched controls. Some high C3 levels, determined immunochemically, were associated with low hemolytic levels of C3, which were attributed to C3 cleavage detected by immunoelectrophoresis. Based on our survival data, ICS, which were significantly different in 20 patients now decreased when compared to those of other patients, are very valuable in the prognosis of neuroblastoma.

Disulfide-linked gamma delta T cell antigen receptors expressed on T cells derived from patients with primary immunodeficiency disorders.

T cell lines or clones from two patients, one with a partial DiGeorge syndrome and one with severe common variable immunodeficiency expressed disulfide-linked gamma delta T cell antigen receptor (TCR) comprised of a gamma-chain polypeptide of 40-43 kD, and a delta-chain polypeptide of 37-40 kD. This gamma delta TCR appears to be similar to that found on T cell clones, and lines derived from peripheral blood lymphocytes from normal donors. Previous studies have shown that T cell lines derived from the peripheral blood of patients with immunodeficiency disorders express non-disulfide-linked gamma delta TCR. In contrast to the latter and coincident with findings in the present study, the vast majority of T cell lines and clones derived from the peripheral blood of normal donors express disulfide-linked gamma delta TCR.

The glomerular mesangium. II. Studies of macromolecular uptake in nephrotoxic nephritis in rats.

These studies were designed to explore the effects of nephrotoxic serum (NTS) in rats on the uptake and processing by the glomerular mesangium of intravenously administered protein macromolecules (radiolabeled aggregated human IgG, [125I]AHIgG). Measurements of [125I]AHIgG levels in preparations of isolated glomeruli correlated well with qualitative estimates of glomerular IgG deposition seen by immunofluorescent microscopy. Rats given 2 ml NTS received 25 mg/100 g body wt [125I]AHIgG 48 h later and were sacrificed at varying time intervals thereafter. NTS-treated animals demonstrated a marked increase in glomerular uptake of [125I]AHIgG as compared with concurrent controls but a normal ability to clear [125I]AHIgG from the mesangium over 72 hr. Animals given 1.0, 0.5, and 0.25 ml NTS had neither proteinuria nor significant light microscopic changes, yet had increased uptake of [125I]AHIgG of 4.4, 3.0, and 2.1 times control values, respectively at 8 h after the infusion of aggregates. Rats given 1 ml NTS and 12.5, 6.25, and 3.125 mg [125I]AHIgG/100 g body wt had increased glomerular uptake at 8 h, but there was, within both NTS and control groups, a disproportionate decrease in uptake at lower [125I]AHIgG doses. Rats given cobra venom factor (CoF) followed by a NTS shown to be complement dependent (proteinuria reduced by prior complement depletion with CoF) had less mesangial [125I]AHIgG uptake than NTS controls but greater uptake compared with normal controls. CoF itself had no effect on mesangial or reticuloendothelial system [125I]AHIgG uptake. These studies demonstrate a striking change in glomerular mesangial activity after fixation of nephrotoxic antibodies to the glomerular basement membrane, even in the absence of proteinuria and suggest that subtle alterations of the filtration surface can influence mesangial function. The effect of NTS on the mesangium is due, in large part, to the glomerular injury and proteinuria which nephrotoxic antibodies produce.

Complete absence of the third component of complement in man.

A 4-yr-old female patient who has recurrent infections with encapsulated bacteria and gramnegative organisms was found to have a complete absence of total hemolytic complement and C3. Total hemolytic complement was reconstituted by the addition of functionally pure C3. With the exception of a moderately reduced homolytic C4, all other C components, measured homolytically and by radial immunodiffusion, were present in normal amounts. By Ouchterlong analysis, the patient's serum contained C3b inactivator and properdin but no antigenic C3. Activation of the alternate pathway was examined by purified cobra venom factor (CVF) and inulin. Neither of these substances led to activation of properdin factor B to B. On addition of partially purified Cordis C3, in four out of four instances and with different preparations of Cordis C3, activation of factor B to B occurred in the inulin-serum-C3 mixture. In contrast, activation of factor B to B occurred only once out of four times with CVF-serum-C3 mixtures. Immune adherence was found to be normal in the patient's serum and could be removed by anti-C4 antiserum of hydrazine treatment. A marked opsonic defect was present against Escherichia coli. Serum bactericidal activity against a rough strain of E. coli was also defective. The ability to mobilize an infalmmatory response was examined by Rebuck skin window technique. A delay in neutrophil migration occurred until the 6th h. In vitro lymphocyte transformation and serum immunoglobulins were normal. The proportion of peripheral blood T cells forming spontaneous sheep erythrocyte rosettes and the percentage of B cells forming EAC rosettes by the C3 receptor were normal. The significance of the absence of C3 in our patient is emphasized by the increased number of infections with encapsulated bacteria and the decreased functional biological activities of the C system, important in host defense mechanism(s).

Bovine antigens and the formation of circulating immune complexes in selective immunoglobulin A deficiency.

We have shown that levels of circulating immune complexes are closely associated with the presence of precipitating antibodies to bovine milk proteins in individuals with selective immunoglobin (Ig)A deficiency. To test whether milk proteins are involved in immune complex formation, sera of seven IgA-deficient individuals were studied for the appearance of complexes after milk ingestion. In three of the seven, an initial fall in the level of complexes was followed by an increasing value, which peaked at 120-150 min. In another three, there was a tendency toward the formation of two peaks of complexes, the first at 30-60 min and the second at 120-150 min after drinking milk. One subject, who had had recent treatment for two separate neoplasms, had a steady level of complexes that did not change during the course of this test. After drinking milk, the molecular weight of the complexes found in the sera of one individual at the start of the milk test fell from >19S to 7-11S, and in vitro additions of progressively increasing amounts of a mixture of milk proteins or bovine gamma globulin, to sera that contained complexes produced a progressive reduction in the level of complexes detectable. We conclude that the circulating immune complexes found in some patients who lack IgA contain bovine milk proteins and that periodic fluctuation of the molecular weight of such complexes, depending upon antigen ingestion, appears likely. It remains uncertain what effect the chronic circulation of complexes has upon the clinical state of this group of patients.

C2 deficiency. Development of lupus erythematosus.

The study of serum from a patient with C2 deficiency is described. The patient had an episode of pneumococcal meningitis at 5 mo of age with seizures and transient hemiparesis and apparent purpuric skin lesions. He was first admitted to the University of Minnesota Hospitals at 10 yr of age following the discovery of proteinuria accidentally by his mother. Since then he has been admitted repeatedly to this hospital with numerous clinical findings including arthralgia, recurrent abdominal pain, proteinuria, membranous nephropathy, malar butterfly rash, seizures, personality aberrations, and recurrent fever. In June 1971, the patient developed positive DNA and DNP antibodies and positive LE cells. When the C profile was studied before and after recognition of lupus, C1q, C1s, and C4 dropped. C3 levels were elevated as were C5, C6, and C7, C3 proactivator had been reduced in the patient even before he developed lupus. Also because of a traumatic renal biopsy leading to a perirenal hematoma, he required surgery and a blood transfusion. 1 h after blood transfusion, a C2 titer of 23 hemolytic units was detected. Almost immediately levels of C3, C5, C6, and C7 dropped, C8 and C9 remained elevated. The addition of C2 from normal blood permitted dramatic activation of C3. These findings support the view that the rare deficiency in production of C2 predisposes to serious susceptibility to infection, vascular and mesenchymal disease as well as to renal disease and a lupus syndrome.

The complement system in bullous pemphigoid. I. Complement and component levels in sera and blister fluids.

Compared with other serum and blister fluid proteins, total hemolytic complement was reduced in the blister fluid of six serologically positive bullous pemphigold patients while four serologically negative cases had blister fluid complement levels closely approaching the serum levels. Except for pemphigus vulgaris blisters. blister fluids from most patients with other bullous diseases and experimentally induced blisters had blister fluid complement levels more closely approaching the serum levels. With the exception of the two terminal components. C8 and C9, individual components of the complement sequence were also reduced in the blister fluids of the six bullous pemphigold patients with circulating basement membrane zone antibodies. On the other hand, transferrin and IgG levels of these same six serologically positive blister fluids closely approached the corresponding serum levels. Conversion of C3 proactivator was also demonstrable in the serologically positive bullous pemphigoid blister fluids, but not in the corresponding sera. Our studies, therefore, are suggestive of local activation of the complement sequence, by both the classical and alternate pathways, in blisters of serologically positive bullous pemphigold patients.

Antigens and antibodies cross-reactive to the murine mammary tumor virus in human breast cyst fluids.

Human breast cyst fluids were shown to contain low concentrations of IgA (15-78 micrograms/ml) and IgG (33-145 micrograms/ml). The IgA:IgG ratios in individual breast cyst fluids ranged from 1:0.6 to 1:4. These levels are considerably higher than their ratio in serum (1:7). IgA from 33% of the 40 fluids examined, and IgG from 10% of the fluids, reacted with the murine mammary tumor virus (MuMTV). The reactivity was detected by an enzyme-linked immunosorbent assay that measures antibody binding to both the envelope glycoprotein and core protein of the virus. In a second series of experiments. IgA from 28% of 40 breast cyst fluids reacted only with MuMTV while IgA from 30% of the fluids was reactive with both MuMTV and the Rauscher murine leukemia virus. Antigen reactive with antiserum to the 28,000-dalton MuMTV core protein (p28), was also identified in a 165,000-g pellet fraction from breast cyst fluids. In individual fluids, the extent of IgA binding to MuMTV was positively correlated (P less than or equal to 0.01) with the binding of anti-p28 antibody to the pellet of the breast cyst fluid. Fractions with the buoyant density of retroviruses (1.16-1.18 g/ml) or their cores (1.21-1.25 g/ml) were isolated from breast cyst fluids. These fractions contained a DNA polymerase capable of utilizing the reverse transcriptase-specific template, dG12-18 x poly rCm. In addition, they reacted with antiserum to MuMTV p 28 but not with antiserum to the 30,000-dalton Rauscher murine leukemia virus core protein.

Sperm-related antigens, antibodies, and circulating immune complexes in sera of recently vasectomized men.

Sera from 35 men were collected before and at timed intervals subsequent to vasectomy and examined for the presence of (a) antibody reactive with human spermatozoa, (b) sperm-related antigen, and (c) circulating immune complexes (CIC). Fewer than 10% of the men examined were ever positive for antisperm antibodies. However, sperm-related antigens were elevated in the sera of 18, 18, and 26% of the mean at 2 wk, 2 mo, and 4 mo postvasectomy, respectively. CIC were detected in the sera of some vasectomized men by three different assays. The CIC in patients' sera were precipitated with polyethylene glycol, dissociated, and the individual CIC components identified by an enzyme-linked immunosorbent assay. Most, but not all, of the CIC contained antigen reactive with antisperm immunoglobulin (Ig)G and some also contained complement components C3 and/or Clq. IgA was identified in some of the CIC positive for IgG and sperm antigen and two men had IgM-containing CIC. Analysis of the CIC by sucrose gradient centrifugation revealed them to be heterogeneous in size.

C1r deficiency: an inborn error associated with cutaneous and renal disease.

The studies of sera from two siblings with C1r deficiency are described. The brother (18 yr old) has shown clinical manifestations resembling lupus erythematosus for 5 yr, and the sister (24 yr old) has had arthralgia and recurrent episodes of rhinobronchitis since early childhood. Three siblings have died: one brother died at age 12 with symptoms similar to the disease of the male patient studied here, and two other siblings died in infancy, probably from infection. The low hemolytic C1 activity of the patients could be restored by the addition of purified C1r to their sera. Bactericidal activity and immune adherence were found to be impaired. When alternate pathways of the complement system were studied, both sera permitted activation of terminal components with endotoxin and cobra venom factor. These findings support the view that an alternate pathway for activation of the terminal portion of the complement cascade exists which does not utilize the conventional pathway operating through the usual early components.

The association of respiratory infection, recurrent hematuria, and focal glomerulonephritis with activation of the complement system in the cold.

The study of the activation of C3, C5, and C7 associated with the conversion of C3 in the serum of a 9-yr old girl after incubation at 0 degrees C for 6-8 h without utilization of C1, C4, and C2 is described. The patient has upper respiratory infections associated with recurrent gross hematuria, focal glomerulonephritis, and transient renal insufficiency. Histological lesions demonstrated the presence of B1c globulin IgA and properdin in the glomeruli. The activation of complement (C) in the cold requires the patient's IgA. Removal of IgA from the serum by immunoadsorption prevents activation and conversion of C3. Bactericidal and phagocytic activity is also impaired after incubation. C3 proactivator (C3PA) level is reduced before and after incubation. Properdin level drops after incubation. These findings suggest that the activation of C3 which is demonstrable in vitro may be a continuous process in vivo.

Genetic control of the eighth component of complement.

Using isoelectric focusing in polyacrylamide gel and a hemolytic assay for development of patterns, extensive, structural polymorphism in human C8 has been delineated. Two alleles, C8A and C8B, have been identified in orientals, with gene frequencies of 0.655 and 0.345. In blacks, what appears to be a third common allele was found, so that frequencies were 0.692, 0.259, and 0.049 for C8A, C8B, and C8A1. In whites, C8A1 was rare with a frequency of 0.003, and frequencies for C8A and C8B were 0.649 and 0.349. Inheritance was autosomal codominant in family studies and the distribution of types in random unrelated populations fit the Hardy-Weinberg equilibrium in all groups. C8 allotypes have been determined for two previously studied families, each with a homozygous C8-deficient propositus. This study suggests that C8 deficiency is a silent or null allele of the C8 structural locus, and that half normal levels of C8 cannot be used as a single criterion for the establishment of heterozygous C8 deficiency. C8 allotypes, as well as 18 other autosomal markers, were also determined for 24 families. The C8 structural locus is not closely linked to these markers, including the human histocompatibility loci complex.

Calories, parity, and prolactin influence mammary epithelial kinetics and differentiation and alter mouse mammary tumor risk.

Reduced calorie intake (RCI) suppresses mouse mammary tumor virus (MMTV) transcription and reduces mammary tumor (MT) incidence in C3H/Ou mice. Since efficient retroviral expression requires cell division, we investigated whether the suppression of MMTV and MT by RCI reflects changes in mammary histogenesis and lowered epithelial kinetics. Prolactin (PRL) augments MMTV transcription. Since PRL levels may be lowered by RCI, we evaluated whether lowered PRL in ad libitum-fed mice alters mammary histogenesis and MT incidence in a manner comparable to RCI. Pregnancy augments MMTV transcription. Hence, we also determined the effect of parity on mammary histogenesis, kinetics, and MT risk. One hundred thirty-five C3H/Ou mice were fed ad libitum or a RCI level and separated into six experimental groups. Twenty ad libitum-fed mice were injected with a dopaminomimetic to lower PRL, and 20 RCI mice were engrafted with adenohypophyses to elevate PRL. Twenty-seven ad libitum-fed mice and twenty-eight RCI mice experienced a single parturition. RCI protected nulliparous (P = 0.0001) and parous mice (P = 0.005) from MT development. Reduced calories or lowered PRL with ad libitum feeding similarly influenced mammary histogenesis, kinetics and MT risk (P > 0.5). Mammary glands of RCI mice or of ad libitum-fed mice with lowered PRL were histologically comparable and principally ductular with a low DNA-labeling index (DNA-LI) (P < 0.001). In contrast, the parenchyma of ad libitum-fed mice or of RCI mice with elevated PRL had exuberant alveoli formation, an elevated DNA-LI (P < 0.001), and preneoplastic lesions. Parity did not change the elevated DNA-LI and MT risk of ad libitum-fed mice but increased the mammary DNA-LI (P < 0.001) and MT incidence (P = 0.01) of RCI mice. Prevention of mammary tumorigenesis in C3H/Ou mice by RCI may result from modulated serum PRL activity and reduced mammary epithelial kinetics which suppress MMTV transcription and minimize the risk of activating protooncogenes.

Calorie intake during mammary development influences cancer risk: lasting inhibition of C3H/HeOu mammary tumorigenesis by peripubertal calorie restriction.

To test for a relationship between peripubertal calorie intake, mammary development, and tumorigenesis, weanling C3H/HeOu mice were separated into 3 groups: fed diet either ad libitum (AL) and designated group AL (n = 60); fed a similar, calorie-restricted (CR) diet only during mammary development when 4-12 weeks old and then subsequently fed ad libitum when > or = 13 weeks old (group CR4-12, n = 24); or continuously calorie restricted (group CR, n = 60). Eight weeks of peripubertal calorie restriction provided CR4-12 mice with lasting protection from mammary tumorigenesis (P = 0.004) and lowered cumulative tumor incidence by 33% compared to AL mice. Sustained calorie restriction of group CR mice further reduced mammary tumor incidence compared to both AL (P = 0.000001) and CR4-12 mice (P = 0.009). Calorie intake significantly influenced mammary development and cellular proliferation. Compared to the mammary development of AL mice, calorie restriction reduced the diameter of ductal end buds (189 microns compared to 146 microns; P < 0.01), lowered the end bud [3H]thymidine labeling index from > or = 20 to < or = 13% (P < 0.001), delayed end bud migration and mammary glandular growth (P < 0.01), reduced alveolar budding (P < 0.001), reduced the proportion of alveoli containing at least one [3H]thymidine labeled cell from > or = 50 to < or = 22% (P < 0.001), and lowered the alveolar [3H]thymidine labeling index of labeled alveoli from > or = 14 to < or = 7% (P < 0.001). These findings link peripubertal calorie intake, mammary development, and carcinogenic risk, and show that the abrogation of mammary tumorigenesis by calorie restriction is partially attributable to influences on mammary development.