Antiglobulin Antibodies Research Articles

Abstract OT1-01-07: nab-paclitaxel (nab-P) plus nivolumab (Nivo) in human epidermal growth factor receptor 2 (HER2)–negative recurrent metastatic breast cancer (MBC)

Abstract Background: Nivo is an inhibitory antibody against programmed death receptor-1 (PD-1), a regulator of antitumor immunity. Nivo is approved for treatment of unresectable or metastatic melanoma and disease progression (PD) following ipilimumab or, in BRAF V600 mutation–positive melanoma, following a BRAF inhibitor, and for metastatic squamous non-small cell lung cancer (NSCLC) following PD during or after platinum-based chemotherapy. Nivo and other immune checkpoint inhibitors are also being investigated in other tumor-types. nab-P is a novel taxane formulation and does not require prophylaxis with immunosuppressive steroids. It has demonstrated superior efficacy over control regimens in phase III studies of MBC, pancreatic cancer, and NSCLC. This open-label, 6-arm, multicenter phase I trial will evaluate the safety of Nivo with nab-P in 3 cancer types (2 arms/disease): MBC, advanced NSCLC (+ carboplatin), and advanced pancreatic cancer (± gemcitabine). The study design for the MBC portion is described below. Methods: Eligibility criteria include histologically/cytologically confirmed HER2-negative MBC; 1 prior chemotherapy for MBC, including an anthracycline unless clinically contraindicated; no relapse < 12 months after taxane adjuvant therapy; measurable disease by RECIST v1.1; ECOG performance status 0-1; adequate organ function; and preexisting peripheral neuropathy grade < 2. Patients (pts) with MBC will be treated in 2 arms: nab-P 100 mg/m2 on days 1, 8, and 15 of each 28-day cycle plus Nivo 3 mg/kg on days 1 and 15 starting at cycle 3 or nab-P 260 mg/m2 on day 1 of each 21-day cycle plus Nivo 5 mg/kg on day 15 starting at cycle 3. Pts will be treated until PD or allowed to continue treatment beyond RECIST v1.1–defined PD if they continue to meet study eligibility; do not have rapid PD or clinical deterioration or unacceptable toxicities; and can benefit from continuation of study treatment in the treating physician's opinion and will not delay an imminent intervention to prevent serious complications of PD. The primary endpoints of the study are the number of pts with dose-limiting toxicities (DLTs) in each treatment arm (part 1) and the percentage of pts with grade 3/4 treatment-emergent adverse events (TEAEs) or treatment discontinuation due to a TEAE (parts 1 and 2). Part 1 of the study will assess whether the starting dose of Nivo is deemed safe (≤ 1 DLT in 6 pts); otherwise, the Nivo dose will be de-escalated and assessed in a new cohort at the next lower dose level. The Nivo dose in combination with nab-P deemed safe in a treatment arm may be further assessed in part 2 of the study, with enrollment expanded to an additional ∼ 14 pts/arm (total of 20 Nivo-treated pts/arm). Secondary study endpoints include TEAEs leading to dose reduction, delay, interruption, or treatment discontinuation; progression-free survival; overall survival; disease control rate; overall response rate; and duration of response (per RECIST v1.1). Exploratory endpoints include tumor-associated PD-L1 expression, modulation of immune activation in the tumor and peripheral blood in response to Nivo treatment, Nivo serum levels, and development of anti-globulin antibodies. ClinicalTrials.gov identifier NCT02309177. Citation Format: Waterhouse D, Gutierrez M, Bekaii-Saab T, DeRosa W, Wainberg Z, George B, Duval Fraser C, Ko A, Pierce DW, Stergiopoulos S, Soliman H. nab-paclitaxel (nab-P) plus nivolumab (Nivo) in human epidermal growth factor receptor 2 (HER2)–negative recurrent metastatic breast cancer (MBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT1-01-07.

Phospholipid Scramblase 1 Binds to the Promoter Region of the Inositol 1,4,5-Triphosphate Receptor Type 1 Gene to Enhance Its Expression

Phospholipid scramblase 1 (PLSCR1) is a multiply palmitoylated, endofacial membrane protein originally identified based on its capacity to promote accelerated transbilayer phospholipid movement in response to Ca(2+). Recent evidence suggests that this protein also participates in cell response to various growth factors and cytokines, influencing myeloid differentiation, tumor growth, and the antiviral activity of interferon. Whereas plasma membrane PLSCR1 was shown to be required for normal recruitment and activation of Src kinase by stimulated cell surface growth factor receptors, PLSCR1 was also found to traffic into the nucleus and to tightly bind to genomic DNA, suggesting a possible additional nuclear function. We now report evidence that PLSCR1 directly binds to the 5'-promoter region of the inositol 1,4,5-triphosphate receptor type 1 gene (IP3R1) to enhance expression of the receptor. Probing a CpG island genomic library with PLSCR1 as bait identified four clones with avidity for PLSCR1, including a 191-bp fragment of the IP3R1 promoter. Using electrophoretic mobility shift and transcription reporter assays, the PLSCR1-binding site in IP3R1 was mapped to residues (-101)GTAACCATGTGGA(-89), and the segment spanning Met(86)-Glu(118) in PLSCR1 was identified to mediate its transcriptional activity. The significance of this interaction between PLSCR1 and IP3R1 in situ was confirmed by comparing levels of IP3R1 mRNA and protein in matched cells that either expressed or were deficient in PLSCR1. These data suggest that in addition to its role at the plasma membrane, effects of PLSCR1 on cell proliferative and maturational responses may also relate to alterations in expression of cellular IP3 receptors.

Determination of IgG and IgM levels in serum by rocket immunoelectrophoresis using yolk antibodies from immunized chickens

A method of rocket immunoelectrophoresis based on the use of avian antiglobulin antibodies is described which permits the quantitation of IgG and IgM in human serum. The electrophoretic mobility of avian yolk immunoglobulin (IgY) and mammalian IgG is sufficiently different to obviate the need to chemically modify the antibody, for example by carbamylation. Rockets are obtained in agarose within 3–6 h in 2-( N-morpholino) ethane sulfonic acid buffer at pH 5.5 or 6.0. Avian globulins specific for human IgG and IgM were obtained easily and in large quantities from the egg yolk of hyperimmunized laying hens. The technique is also applicable to quantitation of immunoglobulins in various animal sera.

Assessment of complement binding by anti-D and anti-M antibodies employing labelled antiglobulin antibodies.

The presence of small amounts of C3d on freshly obtained normal red blood cells (RBC) was demonstrated by a radio-labelled antiglobulin technique; this increased 1.5-fold after incubation in fresh normal serum. No significant further increase in bound C3d was demonstrated for cDE/cDE, CDe/CDe, -D-/-D- RBC maximally sensitized with any of three potent anti-D antibodies. When anti-c, anti-D and anti-E antibodies were used in combination to sensitize cDE/cDE RBC, bound C3d increased by approximately 22%; however, a similar increase occurred with cde/cde RBC and was therefore considered non-Rh specific. Umbilical cord RBC from four infants severely affected by anti-D haemolytic disease of the newborn did not exhibit more bound C3d than cord RBC from normal controls. One serum containing IgM anti-M bound a small amount of complement in vitro; two IgG anti-M sera did not.

Two-stage rocket-electrophoresis on cellulose acetate films

The method of two-stage rocket electrophoresis on gelatinized cellulose acetate film (Cellogel) was developed. The method is based on electroimmunodiffusion detection of antigen on cellulose acetate films containing monospecific antiserum of a test system, followed by detection of precipitation bands with the appearance of stained “rockets” if the reaction takes place in the visible zone, or with further treatment of cellulose acetate strips containing invisible precipitates with antiglobulin antibodies, complement, or a combination of both. The sensitivity of the method is increased to 30–60 ng/ml in the visible zone of reaction by simultaneously reducing the concentration of antibodies and increasing the absolute amount of antigen for electrophoresis to 50–100 λl. Details of the method were worked out for human α-fetoprotein.

Immunological Studies in Psoriasis

The mean values of serum immunoglobulins A, G, M and D levels in 42 psoriasis patients showed no significant difference from those of control subjects. Analysis of the same sera for the presence of antiglobulin antibodies also yielded negative results. Antinuclear factor could be demonstrated in only 4.8% of the cases. The possibility that serum antiglobulins get drained into the lesions making their detection in the serum difficult, is supported by our preliminary findings of focal deposition in the stratum corneum of immune complexes of IgG, IgM and complement in tissue sections of psoriatic lesions from these patients.

A highly sensitive electroimmunodiffusion method of antigen detection on cellulose acetate films

A highly sensitive electroimmunodiffusion method of detecting antigens on cellulose acetate films is suggested. There are three stages: concentration of the antigen in a discontinuous buffer system on cellulose acetate films; detection of the antigen on the same films by immunodiffusion using a standard test systems; staining the washed films with a protein stain to detect precipitation bands if the reaction takes place in the visible zone, or further treatment by “growing” precipitates with antiglobulin antibodies or by autoradiography. The method can detect nanogram amounts of α-fetoprotein and can be used to discover antigens of different molecular weight and electrophoretic mobility.

The C1q Binding Test for Soluble Immune Complexes: Clinical Correlations Obtained in Patients With Cancer 2

Sera from 134 selected patients with various types of cancer were tested for soluble antigen-antibody complexes by the C1q binding method. Sera from 85 healthy blood bank donors served as normal controls. C1q binding activity (C1q BA) values above the 95th percentile for healthy subjects were found in 83% of sera from patients with neoplastic diseases. The incidence of abnormal C1q BA values among patients with malignant melanoma was 83%, with breast cancer 74%, with colon cancer 75%, with lung cancer 88%, with leukemia and lymphoma 85%, and with miscellaneous tumors 94%. High C1q BA values were found most frequently in sera of patients who had been diagnosed relatively recently (within 5 mo) and who had evident residual disease after surgical treatment. Recurrence or progression of tumor growth occurred significantly more frequently in lung cancer patients with high C1q BA. DNA was not detected in cancer patients' sera and treatment with DNase did not decrease in C1q BA. C1q BA in sera could not be explained by the presence of antiglobulin antibodies. Sucrose density gradient ultracentrifugation studies of the serum C1q BA in 4 cancer patients showed that the major binding activity was found between 19S and 7S.

Renal localization of antiglobulins in glomerulonephritis and after renal transplantation

Localization of fluorescein-conjugated heat-aggregated IgG (FA IgG) was demonstrated by immunofluorescence in renal glomeruli of 16 of 69 patients with glomerulonephritis. FA IgG bound more frequently in kidney biopsies from patients with diffuse glomerulonephritis and depressed renal function, and localized selectively in glomeruli that contained heavy deposits of IgM, C3, and C4. The factors that caused FA IgG to bind were specifically reactive with the Fc piece of the IgG molecule and were resistant to 56 degrees C heat for 30 minutes. Localization of FA IgG in the kidney did not correlate with the presence of soluble immune complexes or detectable antiglobulin antibodies in the sera. Binding of FA IgG was also seen in glomeruli and arteries of 18 of 21 kidney allografts studied at the time of impending rejection. But the factors responsible for binding FA IgG in the allografts were heat labile and thus could have been C1q. Although the role of these "antiglobulins" in the immunobiology of glomerulonephritis remains unknown, the fact that they occurred mainly in patients with relatively severe glomerular injury suggests that they could play some part in promoting renal glomerular injury.

Studies on vasculitis. VI. Antiglobulins or rheumatoid-like factors in cutaneous vasculitis lesions detected by an improved immunofluorescence procedure.

Human cutaneous vasculitis lesions tend to be chronic in contrast to ephemeral experimental Arthus responses. Human lesions were examined for fixed antiglobulin antibodies, as occur in rheumatoid joint synovia, which could bind globulins to form tissue-damaging complexes and perpetuate the inflammation. The immunofluorescence procedure was modified by pretreating sections with 5% bovine serum albumin, and by using fluorescein conjugates of F(ab)2 portions of antibodies to avoid the antigen-nonspecific binding that sometimes occurs in inflamed or necrotic tissue. Previous findings of immunoglobulins in lesions were confirmed by the more discriminating technique. IgG and IgM was present more frequently in lesions with mainly mononuclear cell changes, than in those with mainly neutrophil changes, leucocytoclastic or necrotizing vasculitis. Fab occurred in some lesions without Fc of IgG or IgM. Three lesions containing IgM or IgM and IgG, specifically bound heat-aggregated whole IgG or Fc of IgG. None bound IgM. The results indicate that the immunoglobulin in some cutaneous vasculitis lesions is locally formed or fixed antiglobulin, which will bind aggregated IgG possibly forming complexes perpetuating the lesion.

Immunologic studies on the pathogenesis of Raynaud's phenomenon in vibration disease.

These examinations were designed to explain the role of immunologic reactivity in the pathogenesis of Raynaud's phenomenon due to vibration. Patients were divided into 3 groups: 81 with positive and 50 with negative Raynaud's phenomenon in vibration disease, and 37 with Raynaud's disease in patients not exposed to mechanical vibration. In the Raynaud's-positive group, increase of beta globulins, gamma globulins, and antiglobulin antibodies were found. The cold hemagglutinin reaction was detected more often in the Raynaud's-positive, than in the Raynaud's-negative group. IgG serum concentration in persons with prodromal symptoms and IgG immunoglobulins in the developed form of vibration disease was significantly increased. The role of the immunologic basis of Raynaud's phenomenon due to vibration was taken into consideration.

Technique d'hémagglutination passive inverse appliquée à la détection de l'antigène HBs

In this report we present an evaluation of the sensitivity, specificity and ability to detect HBs Ag carriers of a new reversed passive hemagglutination test, using immunochemically purified chimpanzee anti HBs bound to stabilized human erythrocytes. The method was shown to have a sensitivity equal (within one two fold dilution) to that of the Ausria I 125 ratio immuno assay, and in a double blind comparison detected essentially the same number of Hbs Ag containing specimens among volunteer blood donors. The method therefore provides an economical method for the third generation testing of blood donors. The methodology which has been described incorporates a definitive specificity test in which serum drawn before and after immunization of chimpanzees with purified HBs Ag is compared for its ability to neutralize the hemagglutination reaction. The use of serum from the same animal for this purpose avoids the theoretical possibility that antiglobulin antibodies directed at subclass determinants such as Gm of Inv could be differentially inhibited due to possible subclass differences in the blocking sera employed. A reliable test for specificity of HBs Ag screening results is essential to avoid false notification of donors that they are carriers of hepatitis B virus.

Further evaluation of a latex agglutination test for detection of hepatitis B antigen.

A latex test for detection of Hepatitis B Antigen (HBAg) has been demonstrated to have sensitivity equivalent to counterelectrophoresis, but an unacceptable frequency of false‐positive results. The false‐positive rate can be reduced to approximately 3 per cent by heating the test serum for 10 minutes at 60 C. The remaining false positives are largely caused by antiglobulin antibodies in the test serum. Although the test is unacceptable for routine HBAg testing in its present form, its speed and simplicity would make it a useful technique for selection of the safer units when fresh blood components must be transfused prior to completion of the more time‐consuming tests.

Antiglobulin Antibodies and Anticomplementary Factors in Hepatitis B Antigenaemia

A study of an apparently healthy population of Southern African Negroes showed that the HB antigen carrier state may predispose these subjects to produce a higher incidence of antiglobulin antibodies and anticomplementary factors. It is presumed that the anticomplementary factors represent excess HB antigen-antibody complexes in a modified form while the antiglobulin antibodies are the host’s humoral response to the modified complexes.

The incidence of antibodies to leukocytes and gammaglobulin IgG and IgA in a population of multitransfused Southern African Negroes.

Posttransfusion samples of serum from Southern African Negroes who suffered mild to severe hypersensitivity reactions possessed more antileukocyte than antiglobulin antibodies to IgG and IgA. On the other hand, multitransfused patients who did not develop adverse transfusion reactions showed a lower incidence of antileukocyte antibodies and a higher incidence of antiglobulin antibodies. The reduced incidence of antiglobulin antibodies found in posttransfusion samples of serum from patients who developed transfusion reactions may reflect in vivo consumption of such antibodies by antigenin‐excess conditions. For the increased incidence of antileukocyte antibodies in the same group of patients the infusion of relatively small amounts of incompatible leukocytes appears to be inadequate to reduce the corresponding antibody levels.

Increasing the specific activity of anti-influenza serum by antiglobulin antibodies and their bivalent F(ab')2-fragments

Increasing the specific activity of anti-influenza serum by antiglobulin antibodies and their bivalent F(ab')2-fragments

Correlation between diagnosis and results of serologic tests for antiglobulin antibodies

A study of 1,320 serum samples for rheumatoid factors and serum agglutinators revealed poor correlation between test results for rheumatoid factors and the presence of rheumatoid arthritis in hospitalized patients with a variety of different diseases. Although the correlation between test results for serum agglutinators and the presence of suppurative infection was good in hospitalized patients, correlation was very poor in less sick ambulatory patients. These studies reveal that correlation of serologic test results with specific disease states may be profoundly altered by severe systemic disease of diverse etiology.

A comparative study of antiglobulin antibodies and residual anti-D between recipients of intramuscular anti-D immunoglobulin and intravenous plasma anti-D.

Abstract. The incidence of ‘specific’ and ‘non‐specific’ antiglobulin antibodies was determined among 648 multiparous females, 213 recipients of intramuscular anti‐D immunoglobulin and 221 recipients of intravenous plasma anti‐D. Results obtained six months after the administration of anti‐D showed that the formation of ‘specific’ anti‐Gm or anti‐Inv was no greater in recipients of anti‐D immunoglobulin or plasma anti‐D than in the controls. The incidence of ‘non‐specific’ antiglobulins increased from an expected 5% among recipients of intravenous plasma anti‐D to almost 25% in mothers given intramuscular anti‐D immunoglobulin. It is suggested that the raised ‘non‐specific’ antiglobulins may be provoked by a residue of aggregated γ‐globulin components which is known to be present in preparations of Cohn fraction II. Six months after the passive administration of Rh antibodies residual anti‐D activity was more often observed in recipients of intramuscular anti‐D immunoglobulin than in recipients of plasma anti‐D.

A Comparative Study of Antiglobulin Antibodies and Residual Anti-D Between Recipients of Intramuscular Anti-D Immunoglobulin and Intravenous Plasma Anti-D

A Comparative Study of Antiglobulin Antibodies and Residual Anti-D Between Recipients of Intramuscular Anti-D Immunoglobulin and Intravenous Plasma Anti-D

Rheumatoid factors in Salmonella and Schistosoma infections.

Abstract Rheumatoid serum factors (anti-globulin antibodies) were studied in chronic salmonella bacteriaemia associated with schistosomiasis, in acute typhoid and paratyphoid fevers, and in schistosomiasis. Rheumatoid factor existed in high frequency and high titre among patients with either chronic or acute salmonella infection, and in high frequency and low titre among patients with schistosomiasis. Following successful treatment of salmonella infections, titres of rheumatoid factor fell. 2-mercaptoethanol consistently eliminated completely the titres of rheumatoid factors, indicating that all were probably IgM. Similar complete elimination of salmonella somatic antigen agglutinins was observed in sera of some patients with either chronic or acute salmonella infection. This association of rheumatoid factors with chronic bacterial and helminthic infections is not unexpected. However, similar results in acute typhoid and paratyphoid fevers necessitate a review of the pathogenesis of IgM anti-globulin antibodies associated with infections.