A small molecular weight factor in aqueous humor acts on C1q to prevent antibody-dependent complement activation

OBJECTIVE: To review the current literature on genes known to affect fertility in the human and mouse. DESIGN: A literature review was performed and key articles were chosen for focus in the areas of genes with effects only on spermatogenesis and oogenesis, with an emphasis on Y-chromosome-encoded gene families and spermatogenesis. In addition, studies describing genes deleted in transgenic mice were incorporated. RESULT(S): Several gene families on the Y chromosome are implicated in spermatogenic failure, but the link between the genetic lesion and the resulting defect is unclear. Many mouse genes involved in repair and DNA damage monitoring have specific effects on gametogenesis in and around meiosis. CONCLUSION(S): Many genes are involved only in gametogenesis, and some of these are beginning to be understood in terms of their functions. An even larger number of genes is required for gametogenesis, and other functions and mouse models give insights important for human disease.     

Expression of markers of platelet activation and the interpatient variation in response to abciximab

Our study concerns the biological effects of abciximab (c7E3 Fab, ReoPro), a powerful new antiplatelet drug that blocks glycoprotein (GP) IIb-IIIa complexes. Samples were examined from 6 patients with coronary artery disease who received a bolus of abciximab followed by a 10- microg/min infusion for at least 18 hours before percutaneous transluminal coronary angioplasty. Inhibition of ADP-induced PA was maximal for 4 patients but partial (79% and 53%) for 2 others during the infusion. Flow cytometry performed with monoclonal antibodies (PAC-1, AP-6, and F26) specific for the "activated" GP IIb-IIIa complex revealed large decreases in the expression of activation markers on platelets during therapy, but these decreases were less marked when inhibition of ADP-induced PA was incomplete. Residual aggregation was seen for all patients during the infusion when TRAP 14-mer peptide or thrombin was the stimulus. Unblocked GP IIb-IIIa complexes were detected on thrombin-stimulated platelets from the patients by immunoelectron microscopy performed using the monoclonal antibody AP-2. Unblocked GP IIb-IIIa complexes were also detected by flow cytometry when platelets preincubated for 1 hour in vitro with abciximab under saturating conditions were (1) incubated with TRAP 14-mer or (2) permeabilized with Triton X-100. In confirming interpatient variation in the platelet response to a standard dose of abciximab, our results also show that an uninhibited internal pool of GP IIb-IIIa complexes may mediate a residual response to strong agonists.     

IgG autoantibodies to complement C1q in pediatric-onset systemic lupus erythematosus

The ribosomal internal transcribed spacer (ITS) region from individual Gyrodactylus specimens was amplified by polymerase chain reaction (PCR). The reaction amplified the entire ITS1-5.8S-ITS2 region of the ribosomal RNA gene cluster using primers that hybridize to the 3' terminus of the small subunit and the 5' terminus of the large subunit ribosomal RNA genes. The PCR products from Gyrodactylus salaris and Gyrodactylus thymalli were cloned and sequenced. The Gyrodactylus 5.8S gene was identified following comparative alignment of the G. salaris sequence and a Schistosoma 5.8S gene sequence. The ITS regions from G. salaris, G. thymalli, Gyrodactylus derjavini, and Gyrodactylus truttae were compared by restriction enzyme analysis and interspecific restriction fragment length polymorphisms were found. Gyrodactylus salaris and G. thymalli restriction fragment sizes were confirmed from sequence data. ITS amplification followed by Sau3AI digestion enables rapid and clear differentiation of G. salaris, G. derjavini, and G. truttae.     

Eccentric exercise augments the cardiovascular response to static exercise

High-force eccentric exercise induces neuromuscular dysfunction and may augment the cardiovascular response to exercise. This investigation sought to determine whether changes in strength and sense of force following high-force eccentric exercise alter heart rate and blood pressure responses during isometric contractions. Subjects (4F,6M) performed 50 maximum resistance eccentric actions with one arm (ECC arm). Contractions at 10% of the ECC arm maximum were held for 7 min on two pre-exercise days. The force output perceived to be the same as 10% of the pre-exercise maximum was determined using a force matching task. This force, 35.6, 27.2, and 21.1% lower on days 1, 3, and 5 post-exercise, was held during isometric contractions on these days, respectively. Despite a lowering of absolute contraction force, heart rate (P < 0.05) and blood pressure (P < 0.001) responses during contractions using the ECC arm were consistently elevated relative to the control arm. However, subjects perceived that they were exerting forces similar to those achieved before eccentric exercise-induced neuromuscular dysfunction. These findings suggest that perceived effort following strength loss induced by mechanically stressful exercise dictates the cardiovascular responses during isometric contractions.     

C1q and systemic lupus erythematosus

In this chapter we review the association between SLE and C1q. In the first part of the chapter we discuss the clinical associations of C1q deficiency, and tabulate the available information in the literature relating to C1q deficiency and autoimmune disease. Other clinical associations of C1q deficiency are then considered, and we mention briefly the association between other genetically determined complement deficiencies and lupus. In the review we explore the relationship between C1q consumption and lupus and we discuss the occurrence of low molecular weight (7S) C1q in lupus, which raises the possibility that increased C1q turnover in the disease may result in unbalanced chain synthesis of the molecule. Anti-C1q antibodies are also strongly associated with severe SLE affecting the kidney, and with hypocomplementaemic urticarial vasculitis, and these associations are also examined. We address the question of how C1q deficiency may cause SLE, discussing the possibility that this may be due to abnormalities of immune complex processing, which have been well characterised in a umber of different human models. There is clear evidence that immune complex processing is abnormal in patients with hypocomplementaemia, and this is compatible with the hypothesis that ineffective immune complex clearance could cause tissue injury, and this may in turn stimulate an autoantibody response. We have also considered the possibility that C1q-C1q receptor interactions are critical in the regulation of apoptosis, and we explore the hypothesis that dysregulation of apoptosis could explain important features in the development of autoimmune disease associated with C1q deficiency. An abnormally high rate of apoptosis, or defective clearance of apoptotic cells, could promote the accumulation of abnormal cellular products that might drive an autoimmune response. Anti-C1q antibodies have been described in a number of murine models of lupus, and these are also briefly discussed. We focus on the recently developed C1q "knockout" mice, which have been developed in our laboratory. Amongst the C1q deficient mice of a mixed genetic background high titres of antinuclear antibodies were detected in approximately half the animals, and around 25% of the mice, aged eight months had evidence of a glomerulonephritis with immune deposits. Large numbers of apoptotic bodies were also present in diseased glomeruli, and this supports the hypothesis that C1q may have a critical role to play in the physiological clearance of apoptotic cells.     

Caffeine abstinence augments the systolic blood pressure response to adenosine in humans

Blood pressure and heart rate responses to adenosine infusion (35, 70, and 140 microg/kg/min, intravenously) were studied in 7 healthy men after 6, 30, 78, 150, and 318 hours of abstinence from regular caffeine use. The finding that caffeine abstinence augmented the systolic pressor response (from -1 +/- 2 mm Hg at 6 hours to +9 +/- 2 mm Hg at 318 hours; p = 0.01) but not the tachycardic response to adenosine has implications for current clinical and research applications of this purine.     

A novel serum protein similar to C1q, produced exclusively in adipocytes

We describe a novel 30-kDa secretory protein, Acrp30 (adipocyte complement-related protein of 30 kDa), that is made exclusively in adipocytes and whose mRNA is induced over 100-fold during adipocyte differentiation. Acrp30 is structurally similar to complement factor C1q and to a hibernation-specific protein isolated from the plasma of Siberian chipmunks; it forms large homo-oligomers that undergo a series of post-translational modifications. Like adipsin, secretion of Acrp30 is enhanced by insulin, and Acrp30 is an abundant serum protein. Acrp30 may be a factor that participates in the delicately balanced system of energy homeostasis involving food intake and carbohydrate and lipid catabolism. Our experiments also further corroborate the existence of an insulin-regulated secretory pathway in adipocytes.     

Evidence that C1q binds specifically to CH2-like immunoglobulin gamma motifs present in the autoantigen calreticulin and interferes with complement activation

Calreticulin (CRT) is located predominantly in the endoplasmic reticulum (ER) of cells, where it functions as a quality control controller of protein folding. However, CRT is also a prevalent autoantigen in patients with systemic lupus erythematosus (SLE), where its release from the cell may arise as a results of dysfunctional apoptosis and inefficient removal of ER vesicles, which are an abundant source of CRT and other autoantigens. Indicative of this is the presence of autoantibodies against CRT in the sera of 40-60% of all SLE patients. Once released into the circulation, CRT might bind directly to C1q and we have suggested that this association may result in a defect in C1q-mediated clearance of antigen-antibody complexes. It has been previously shown that CRT under physiological salt conditions binds to the globular head of C1q. It is known that the globular head region of C1q binds to the CH2 domain in the Fc portion of immunoglobulin gamma (IgG). The N-terminal half of CRT contains a number of short regions of 7-10 amino acids that show sequence similarity to the putative C1q binding region in the CH2 domain of IgG. By use of a series of 92 overlapping CRT synthetic peptides, a number of C1q binding sites on the CRT molecule have been identified, including several containing a CH2-like motif similar to the ExKxKx C1q binding motif found in the CH2 domain of IgG. A number of these peptides were shown to inhibit binding of C1q to IgG and reduce binding of native CRT to C1q. Moreover, several of the peptides were capable of inhibiting the classical pathway of complement activation. These studies have identified specific binding sites on the CRT molecule for C1q and lend support to the hypothesis that interaction of CRT with C1q may interfere with the ability of C1q to associate with immune complexes in autoimmune-related disorders.     

Charge-based binding of complement component C1q to the Alzheimer amyloid beta-peptide

Activation of the classical pathway in Alzheimer's disease derives from the binding of the first protein, subcomponent C1q, to the amyloid beta-peptide (A beta). Analysis of the binding of C1q to A beta by competitive enzyme-linked immunosorbent assay shows that A beta fragments 1-16 and 1-28 but not 12-28 and 17-42 are capable of inhibiting the A beta/C1q interaction, implicating the A beta 1-11 region as the C1q binding site. Binding is also shown to be inhibited by conditions of high ionic strength, suggesting that charged side chains in the A beta 1-11 region are critical to the A beta/c1q interaction. Ultrastructural evidence of binding is provided by platinum replica electron microscopy. Along with a previous demonstration of the 14-26 region of the C1q A chain as the A beta binding site, these findings suggest that attractions between a negative charge cluster in A beta 1-11 and a positive charge cluster in C1qA14-26 mediate the binding of A beta and C1q.     

Siliconized glass versus polypropylene to reduce in vitro platelet activation

BACKGROUND: CSF shunting procedures are generally considered the fundamental therapy of syphilitic hydrocephalus. METHODS: We followed up with CSF analysis and MR imaging a patient with progressive mental and gait disturbances and tetraventricular hydrocephalus due to tertiary syphilis who was treated for 14 days with high dose intravenous penicillin alone. RESULTS: Clinical and CSF abnormalities resolved within a few months, whereas the hydrocephalus disappeared only 30 months after therapy. CONCLUSIONS: Before consideration of a CSF shunting procedure, a trial of high dose intravenous penicillin is warranted for patients with syphilitic hydrocephalus.     

Supplementation of MCF-7 cells with essential fatty acids induces the activation of protein kinase C in response to IGF-1

V(D)J recombination consists of a DNA cleavage reaction catalysed by RAG1 and RAG2, followed by an end-joining reaction that utilizes the cell's double-strand break repair machinery. Genes essential for the end-joining reaction include: XRCC4 encoding a protein of unknown enzymatic function; XRCC5 and XRCC6 encoding 86 and 70 kDa subunits of the Ku autoantigen, a DNA end-binding protein that is also the regulatory subunit of DNA-dependent protein kinase (DNA-PK); and XRCC7 encoding the catalytic subunit (DNA-PKcs) of DNA-PK. Recent progress in understanding the cleavage reaction, coupled with what was previously known about Ku, DNA-PK, and double-strand break repair, provide the foundation for a working model of how V(D)J recombination might be catalysed.     

Evidence of platelet activation in hypertension

To test the hypothesis that platelet activation is present in hypertension, we measured plasma markers beta thromboglobulin and soluble P-selectin in hypertensive patients and normotensive controls. Both markers were raised in the patients (P < 0.05), and in a subgroup of patients, beta thromboglobulin was reduced with successful treatment of hypertension with the ACE inhibitor quinapril. We suggest that reversible platelet activation is present in hypertension. This may be a contributing factor to the link between this risk factor and the development of thrombotic disease such as stroke.     

Transendothelial migration of megakaryocytes in response to stromal cell-derived factor 1 (SDF-1) enhances platelet formation

Cerivastatin sodium, a synthetic and pure enantiomeric 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, is considered effective in the treatment of mild-to-moderate primary hyper-cholesterolemia (total cholesterol < or = 220-259 mg/dL) at a daily dose of 0.15 mg. We compared the efficacy and tolerability of a dosage of 0.3 mg/d with those of a dosage of 0.15 mg/d in patients with severe primary hypercholesterolemia (serum total cholesterol > or = 260 mg/dL). After a minimum of 4 weeks' lead-in with placebo, 73 patients with severe primary hypercholesterolemia were randomly assigned to receive either 0.15 or 0.3 mg of cerivastatin sodium once daily after the evening meal for 12 weeks. In 58 patients, the same drug was continued at a flexible dosage for an additional 36 weeks or longer to assess the long-term efficacy and tolerability of cerivastatin sodium. During the 12-week treatment period, serum total cholesterol levels decreased significantly from baseline in both dosage groups, but the percentage reduction was significantly greater in the 0.3-mg group (range, 24.4% to 25.6%) than in the 0.15-mg group (range, 19.4% to 21.6%). The percentage reduction in levels of low-density lipoprotein cholesterol, triglycerides, and apolipoprotein B and the percentage increase in levels of high-density lipoprotein cholesterol were significantly greater in the 0.3-mg group than in the 0.15-mg group. When the results for the 0.3- and 0.15-mg groups were combined, the percentage of change in serum lipid levels at 48 weeks remained as stable as at 12 weeks. No serious adverse reactions were observed. We concluded that the higher dose of cerivastatin sodium was more effective than the lower dose, with comparable tolerability, in the treatment of patients with severe primary hypercholesterolemia.     

Gender differences in platelet GPIIb-IIIa activation

Gender differences in the development of thrombotic diseases have been described in numerous clinical settings. Enhanced platelet reactivity in both sexes is associated with the development of vascular thromboses. Because activation of platelet GPIIb-IIIa receptors is a central event in thrombus formation, we examined GPIIb-IIIa function in normal male and female volunteers. Using flow cytometry, we quantitated gender differences in the number of binding sites for FITC-labeled fibrinogen (FITC-FGN) and FITC-labeled PAC-1 antibody (FITC-PAC-1). Washed platelets were incubated with either FITC-FGN or FITC-PAC-1 and activated with either ADP or thrombin receptor activating peptide (TRAP) prior to cytometric acquisition of data. The dissociation constant for FITC-FGN was the same in both sexes (approx. 1.6 x 10(-7)M), however, the number of GPIIb-IIIa receptors per platelet capable of binding fibrinogen was significantly greater in women than men in response to 2 microM ADP (16,319 +/- 1871 vs 9669 +/- 1994, p = 0.02), 20 microM ADP (39,951 +/- 4711 vs 25,948 +/- 4953, p = 0.05) and 50 microM TRAP (39,236 +/- 3965 vs 21,848 +/- 4159, p = 0.007). Similarly, the number of GPIIb-IIIa receptors capable of binding PAC-1 in response to ADP and TRAP was 50% to 80% greater in women than men. Binding experiments using specific anti-GPIIb-IIIa monoclonal antibodies (P2 and 10E5), as well as quantitative Western blotting experiments, showed no gender difference in the total number of GPIIb-IIIa molecules expressed. Analysis of data from female subgroups demonstrated an association of GPIIb-IIIa reactivity with menstrual phase. We conclude that GPIIb-IIIa receptors on platelets from premenopausal women are more "activatable" than those on platelets from young men. Variations in the serum concentrations of estrogens and/or progestins may modulate GPIIb-IIIa function.     

Heterogeneity of antibody response to human platelet transfusion

To study the antibody response to human platelet transfusions, nine thrombocytopenia patients with bone marrow failure were given 6 U (3X10(11)) of random platelet concentrates twice a week. Before transfusion, none of the patients had preexisting antibodies detectable with lymphocytotoxicity, platelet aggregation, or capillary leukoagglutination techniques. After receiving 18-78 U of platelets, they became refractory to further transfusions of random platelets and alloantibodies were detectable. Two patterns of antibody response could be identified. In three patients, the sera were not lymphocytotoxic with a panel of standard cells in which all the known HLA antigens in the first and second series were represented at least once. Yet, they caused platelet aggregation with 30, 24, and 60%, respectively, of a donor population studied. The aggregating activities were inhibited by antihuman IgG but not by antihuman IgA or antihuman IgM antiserum. The aggregating antibodies could be absorbed out with donor platelets but not lymphocytes or granulocytes. Antibodies from two of these patients aggregated platelets of their respective siblings matched for both HLA haplotypes. Transfusion of platelets from these two siblings did not increase the platelet count while platelets obtained from aggregation-negative donors did. The sera from the remaining six patients were lymphocytotoxic with 15-100% of the panel of standard cells. They also had aggregating antibodies, which could be absorbed out by both platelets and lymphocytes, suggesting that they were HLA antibodies. These data suggest that the development of platelet-specific antibodies may play an important role in the immunological rejection of isologous platelets, and should be considered in the selection of donors for patients who are refractory to platelets from random donors.     

Immune response in the hamster. VIII. Ig class differences in susceptibility to tolerance induction

A hemolytic plaque assay was used to quantitate the antibody response of the Syrian hamster after immunization with hen egg albumin (HEA). Whereas HEA in complete Freund's adjuvant (HEA-CF) induced a prolonged heterogeneous (IgM, IgG1 and IgG2) antibody response, the response to soluble HEA in saline (HEA-S) differed in that: 1)both the primary and secondary responses were restricted to the IgG1 class; 2)the IgG1 primary response was cyclical with PFC peaks on days 9 and 16; 3)although an anamnestic secondary response was demonstrated, no further augmentation was noted after tertiary and quaternary boosters; 4)the booster response was transient reaching a peak after 48 hr and declining to low levels within 7 days. Adoptive transfer of lymph node cells to irradiated recipients followed by challenge with HEA-CF revealed: 1)that HEA-S-treated donor cells were primed for an IgG1 response because anamnesis was seen 7 days after challenge, yet on day 21, IgG1 PFC were 20-fold less than that of controls; 2)recipients of HEA-S treated cells showed profound suppression of both IgM and IgG2 PFC on days 14 and 21. These studies indicate that soluble antigen induced in hamsters a state of complete tolerance of IgM and IgG2 classes whereas the anamnestic response of the IgG1 class remained intact.     

Flow cytometric assays to detect platelet activation and aggregation in device-implanted calves

Omenn's syndrome is an inherited human combined immunodeficiency condition characterized by the presence of a large population of activated and tissue-infiltrating T cells. Analysis of the TCRB repertoire revealed a highly restricted TCRBV usage in three patients. More strikingly, T cell clones from the three patients expressed TCRB chains with VDJ junction similarities, suggesting a common antigenic specificity. Analysis of the TCRA repertoire in one patient also revealed a restricted TCRAV usage. Finally, analysis of the TCRBV repertoire of tissue-infiltrating T cells in one patient suggested nonrandom tissue migration. These results suggest that the oligoclonal expansion of T cells observed in Omenn's syndrome could be the consequence of autoimmune proliferation generated by a profound defect in lymphocyte development.     

Contributions to maxima in protein kinase C activation

In many lipid systems, the activity of protein kinase C (PKC) exhibits a peak followed by a decline as the mol % of one component is increased. In these systems, an increase in one lipid component is always at the expense of another or accompanied by a change in total lipid concentration. Here we report that in saturated phosphatidylserine (PS)/phosphatidylcholine (PC)/diacylglycerol (DAG) mixtures, increasing PS or DAG at the expense of PC revealed an optimal mol % PS, dependent on mol % DAG, with higher mol % PS diminishing activity. The decrease at high mol % PS is probably not attributable simply to more gel-phase lipid due to the higher melting temperature of saturated PS versus PC because a similar peak in activity occurred in unsaturated lipid systems. Increasing the total lipid concentration at suboptimal mol % PS provided the same activity as higher mol % PS at lower total lipid concentration. However, at optimal mol % PS, activity increased and then decreased as a function of total lipid concentration. PKC autophosphorylation also exhibited an optimum as a function of mol % PS, and increasing the PKC concentration increased the mol % PS at which activity decreased, both for autophosphorylation and for heterologous phosphorylation. Formation of two-dimensional crystals of PKC on lipid monolayers also exhibited a peak as a function of mol % PS, and the unit cell size of the crystals formed shifts from 50 x 50 A at low mol % PS to 75 x 75 A at higher PS. Collectively, these data suggest the existence of optimal lipid compositions for PKC activation, with increased quantity of these domains serving to dilute out enzyme-substrate aggregates and/or enzyme-enzyme aggregates on the lipid surface.     

C1q receptors: regulating specific functions of phagocytic cells

A C1q receptor that upregulates the phagocytic capacity of professional phagocytes, C1qRp, has been identified, and its primary structure determined by cDNA cloning and sequencing. Monoclonal antibodies that immunoprecipitate this 126,000 Mr polypeptide inhibit the enhancement of phagocytosis triggered not only by C1q but also by mannose binding lectin (MBL) and pulmonary surfactant protein A (SPA) providing critical evidence that this polypeptide is a functional receptor or component of the receptor that mediates this enhancement of phagocytosis. The amino acid sequence, deduced from the cloned cDNA coding for this receptor, indicates that this surface glycoprotein receptor is a novel type I membrane protein of 631 amino acid containing a region homologous to C-type lectin carbohydrate recognition domains, 5 EGF-like domains, a single transmembrane domain and a 47 amino acid intracellular domain. Expression of this receptor is limited to cells of myeloid origin, platelets and endothelial cells, consistent with a relatively selective function, and making it an attractive candidate for therapeutic modulation of function. A distinct C1q receptor that triggers superoxide in polymorphonuclear leukocytes has been functionally characterized and designated as C1qRO2-. Thus, the accumulated data that will be summarized here demonstrate that there are at least two C1q receptor/receptor complexes (C1qRp and C1qRO2-), each triggering distinct cellular responses, that multiple C1q receptors can be expressed on the same, as well as on different, cell types, and that at least one C1q receptor, C1qRp, is capable of responding to multiple ligands.