Mg Of Monoclonal Antibody Research Articles

Protective Effects of Monoclonal Antibody to Intercellular Adhesion Molecule-1 in Venous Ischemia-Reperfusion Injury: Experimental Study in Rats

Flaps with venous occlusion have a decreased survival rate compared with arterial occlusion. It seems that several factors are involved in the etiology of total venous occlusion, including free radicals, edema, thrombosis, and reperfusion injury. In the present study, the authors evaluated the blockage of polymorphonuclear leukocyte endothelial adhesion by using a monoclonal antibody to the intercellular adhesion molecule 1 (ICAM-1) ligand to prevent venous ischemia-reperfusion injury in rat epigastric island flaps. A skin flap (3 x 4 cm) supplied by the superficial epigastric artery and vein was harvested unilaterally in 40 male Wistar rats. Total venous occlusion of the skin flap was achieved. Arterial inflow was left intact. Rats were randomly divided into four groups (n = 10). In Group 1; rats were intravenously pretreated with 0.5 ml of 0.9 percent normal saline 15 min before applying a venous clamp, and the flaps were subjected to 6 hr of venous ischemia. In Group 2; rats were intravenously pretreated with 0.05 mg of monoclonal antibody to the intercellular adhesion molecule 1 (0.20 mg/kg) in 0.5 ml of 0.9 percent normal saline 15 min before applying the venous clamp, and the flaps were subjected to venous ischemia as in Group 1. In Group 3; rats were pretreated as in Group 1, and the flaps were subjected to 8 hr of venous ischemia. In Group 4; rats were pretreated as in Group 2, and the flaps were subjected to 8 hr of venous ischemia. The flaps were assessed histologically and by measuring viable and non-viable areas on postoperative day 7. Flap measurements revealed that blocking the action of ICAM-1 IN VIVO by administering monoclonal antibody significantly attenuated ischemic injury after 6 or 8 hr of venous occlusion.

CB.Hep-1 hybridoma growth and antibody production using protein-free medium in a hollow fiber bioreactor.

The protein-free medium TurboDoma HP.1 (THP.1) was used to produce the CB.Hep-1 monoclonal antibody (mAb) in a CP-1000 hollow fiber bioreactor (HFB). This mAb is used for the immunopurification of recombinant hepatitis B surface antigen (rHBsAg), which is included in a vaccine preparation against the Hepatitis B Virus. By using the experimental conditions tested in this work we were able to generate more than 433 mg of IgG in 43 days. The maximum antibody concentration obtained was about 2.4 mg ml(-1)and the IgG production per day was approximately 11 mg of monoclonal antibody, which constitutes a good concentration value in comparison to the results obtained in ascitic fluid, where concentration for this hybridoma was around 3 mg ml(-1). We used different analytical methods to control the quality of mAbs, obtained from the in vitro system. They included affinity constant determination, analysis of N-glycan structures, immunoaffinity chromatography and antigen binding properties. The results obtained suggest that no significant changes occurred in the mean characteristics of the mAb harvested from the bioreactor during the 43 days of cultivation.

IL-4–induced activation of the Stat6 pathway contributes to the suppression of cell-mediated immunity and death in sepsis

Background: Although studies have shown that there is a marked depression in cell-mediated (TH1) immunity after the onset of sepsis, the mechanism by which this occurs remains unknown. In this regard, the TH2 cytokine IL-4 is known to regulate TH1 and TH2 cell responsiveness primarily through the activation of the signal transducer and activation of transcription factor-6 (Stat6) pathway. Methods: We hypothesized that IL-4 may contribute to the suppression of cell-mediated immunity and to death seen in sepsis and that IL-4 may be acting through the Stat6 pathway. To determine this, we induced cecal ligation and puncture (CLP) or sham-CLP in male BALB/c mice. Mice received 2 mg of monoclonal antibody against IL-4 or IgG control at 12 hours after CLP (ie, at the onset of immune suppression). Splenic T cells were then isolated 24 hours after CLP and stimulated with monoclonal antibody to CD3. Cytokine release and Stat6 phosphorylation (activation) were determined. In a separate group of animals, survival was assessed over 10 days. Results: The results indicate that after CLP, T cells are suppressed in their ability to release the TH1 cytokines, IL-2 and IFN-γ. Alternatively, the release of TH2 cytokines IL-10 and IL-4 is markedly increased after CLP. This was associated with an increase in phosphorylated Stat6 protein. In vivo treatment of mice with monoclonal antibody to IL-4 at 12 hours after CLP restores TH1 responsiveness while preventing the increase in TH2 cytokine release and Stat6 phosphorylation. Furthermore, neutralization of IL-4 markedly increased the survival rates in septic animals. Conclusions: Taken together, these data indicate that the TH2 cytokine IL-4 contributes to the suppression of cell-mediated immunity and death associated with polymicrobial sepsis and suggest that IL-4 may be acting through the Stat6 pathway in septic animals. (Surgery 2000;128:133-8.)

Monoclonal antibodies specific for rat relaxin. X. Endogenous relaxin induces changes in the histological characteristics of the rat vagina during the second half of pregnancy.

This study employed morphometric analysis to evaluate changes in the histological characteristics that accompany relaxin-induced growth and softening of the vagina during the second half of rat pregnancy. There were three treatment groups (N = 4/group). Five milligrams of a monoclonal antibody for rat relaxin, designated MCA1, were injected i.v. daily on days 12-21 of gestation to treatment group MCA1. Control groups received either 5 mg of monoclonal antibody for fluorescein (MCAF; monoclonal antibody control) or 0.5 ml PBS (vehicle control). Vaginas were removed on day 22 of pregnancy, fixed in 10% neutral-buffered formalin, and embedded in paraffin. Tissue sections (5 microm) were stained with Gomori's trichrome to visualize collagen, or orcein to visualize elastin. Measurements were performed with a light microscope equipped with a video camera connected to a computer. Within the vaginal stroma, the density of collagen fiber bundles was lower, the length of elastin fibers was shorter, and the cross-sectional area and wall thickness of arteries were greater in relaxin-replete control rats than in relaxin-deficient MCA1-treated rats. These relaxin-induced changes in the stroma appear to account, at least in part, for the hormone's softening effect on the vagina. Within the epithelium, there were approximately 2-fold more basal and mucus-secreting cells in relaxin-replete control rats than in MCA1-treated rats. The relaxin-induced accumulation of epithelial cells appears to contribute to vaginal growth. We conclude that relaxin plays a role in preparing the vagina as well as the cervix for rapid and safe delivery in pregnant rats.

Monoclonal antibody to intercellular adhesion molecule 1 protects skin flaps against ischemia-reperfusion injury: an experimental study in rats.

The purpose of this study was to evaluate the blockage of polymorphonuclear neutrophil endothelial adhesion by using a monoclonal antibody to the intercellular adhesion molecule 1 (ICAM-1) ligand to prevent ischemia-reperfusion injury in rat skin flaps. A skin and subcutaneous tissue flap (3.0 cm x 4.5 cm) supplied by the superficial epigastric artery and vein including the femoral vessels was isolated unilaterally in 45 male Sprague-Dawley rats and clamped for 9 hours (groups II and III) or 12 hours (groups IV and V) of ischemia. Five animals in group I were sham-operated only with 5 minutes of ischemia. Animals in groups II (n = 10) and IV (n = 10) received 0.05 mg of monoclonal antibody to ICAM-1 (0.20 mg/kg) in 0.5 ml of 0.9% normal saline intravenously 15 minutes before reperfusion; those in groups III (n = 10) and V (n = 10) received 0.5 ml of normal saline. The flaps were assessed histologically, by measuring viable and nonviable areas, and by diffuse reflectance spectroscopy to determine the ratio of oxyhemoglobin to deoxyhemoglobin. Flap measurements revealed that the average area of flap survival was 90.6 +/- 12.8 percent in group II and 18.3 +/- 19.6 percent in the control group (III) (p < 0.002). In the animals subjected to 12 hours of ischemia, those treated with monoclonal antibody to ICAM-1 (group IV) were 57.1 +/- 23.1 percent viable, which was significantly greater than the control animals (group V), in which only 0.3 +/- 1.0 percent of the flap was viable. Analysis of the diffuse reflectance spectra showed a hyperemic response during the first 10 minutes after reperfusion in animals treated with monoclonal antibody to ICAM-1. In group III, however, the spectra demonstrated a decreased amount of oxyhemoglobin, indicating decreased reperfusion of the flap after ischemia when compared with group II. Histopathologically, few inflammatory changes could be observed in groups I, II, and the viable areas of group IV. Marked damage was observed in groups III and V. We concluded that treating ischemic skin flaps with monoclonal antibody to ICAM-1 was effective for alleviating reperfusion injury after 9 or 12 hours of warm ischemia. The reactive hyperemic response determined by diffuse reflectance spectroscopy in groups II and IV correlated with areas of flap survival. Antibodies to particular adhesion molecules, such as ICAM-1, have potential clinical utility in that they could be administered, individually or together, to patients immediately before reestablishing perfusion after free-tissue transfer or replantation to block the adverse effects attributed to reperfusion injury.

An approach to integrated antibody production: Coupling of fluidized bed cultivation and fluidized bed adsorption

Continuous culture may be an efficient way of producing proteins which are susceptible to secondary processing in the course of a fermentation process. Short residence times in these systems support the production of correctly assembled proteins by avoiding substrate limitations and product inhibitions and also minimize the contact of sensitive bioproducts with degrading enzymes. Thus products of increased stability and integrity are obtained from continuous processes. The downstream process following continuous culture has to be adapted to the specific conditions of continuous fermentations, e.g. large liquid volumes and diluted process solutions. In this paper an approach is shown how a fluidized bed adsorption as first recovery operation may be coupled directly to a continuous production. Immobilized hybridoma cells are cultivated in porous glass microcarriers in a continuous fluidized bed process, the cell containing harvest is purified by fluidized bed adsorption using an agarose based cation exchange matrix. By this coupled mode of operation the large biomass containing harvest volume resulting from the continuous cultivation may be applied directly to a fluidized chromatographic matrix without prior clarification, leading to a particle free and initially purified product solution of reduced volume. In an experimental setup a bench-scale fluidized bed bioreactor of 25 ml carrier volume was coupled to a fluidized bed adsorption column operated with 300 ml of adsorbent. This configuration yielded up to 20 mg of monoclonal antibody per day in a cell free solution at fourfold concentration and fivefold purification. The process was run for more than three weeks with consistent product output.

Inhibition of bombesin-stimulated acid secretion by immunoneutralization of gastrin in dogs

Bombesin-like peptides stimulate gastrin release and gastric acid secretion. The increase in gastric acid output is thought to be secondary to gastrin release. A monoclonal antibody (MAb) directed specifically to gastrin (MAb 28.2) was used to study the role of circulating gastrin in the regulation of bombesin-stimulated acid secretion in dogs. Seven conscious, fasted dogs with gastric fistulas received intravenous bombesin infusions in fourfold increasing doses from 200 to 3,200 pmol.kg-1.h-1. Each dose was given for 45 min. On separate days, dogs were pretreated with an intravenous infusion of 7 mg of MAb 28.2 or vehicle (0.1% canine serum albumin). Samples of gastric effluent were collected by gravity drainage through the gastric fistula, and acid output was measured by titration of gastric effluent to pH 7.0, using 0.2 N NaOH. Plasma gastrin concentrations were determined by radioimmunoassay. Bombesin infusion produced dose-dependent increases in plasma gastrin concentrations and gastric acid output. Administration of gastrin MAb 28.2 abolished bombesin-stimulated gastric acid output. Immunoneutralization of circulating gastrin in vivo using a gastrin monoclonal antibody in dogs indicates that the acid stimulatory response to bombesin is mediated by gastrin.

Differential Th1 and Th2 cell responses in male and female BALB/c mice infected with coxsackievirus group B type 3

Male and female BALB/c mice differ dramatically in susceptibility to myocarditis subsequent to coxsackievirus B3 (CVB3) infection. CVB3 infection of male mice results in substantial inflammatory cell infiltration of the myocardium, and virus-immune lymphocytes from these animals give predominantly a Th1 cell phenotypic response, as determined by predominant immunoglobulin G2a isotypic antibody production and elevated numbers of gamma interferon and interleukin-2 (IL-2)-producing CD4+ T lymphocytes. Females infected with the same virus give predominantly a Th2 cell phenotypic response, as determined by preferential immunoglobulin G1 antibody isotypic responses and increased precursor frequencies of IL-4- and IL-5-producing CD4+ T cells. Treatment of females with testosterone or males with estradiol prior to infection alters subsequent Th subset differentiation, suggesting that the sex-associated hormones have either a direct or indirect effect on CD4+ lymphocyte responses in this model. Treatment of females with 0.1 mg of monoclonal antibody to IL-4 reduces precursor frequencies of IL-4-producing CD4+ T cells and increases frequencies of gamma interferon-producing cells. This treatment also enhances myocardial inflammation, indicating a correlation between Th1-like cell responses and pathogenicity in CVB3 infection. The Th2-like cell may regulate Th1 cell activation. Adoptive transfer of T lymphocytes from CVB3-infected female mice into male animals suppresses the development of myocarditis in the recipients. Treatment of the female donors with monoclonal antibodies to either CD3, CD4, or IL-4 molecules abrogates suppression.

Effect of orally administered monoclonal antibody on persistent Cryptosporidium parvum infection in scid mice.

Scid mice, persistently infected after exposure to 10(7) Cryptosporidium parvum oocysts, were treated daily for 14 to 17 days with 0.4 mg of monoclonal antibody (mAb) 17.41 administered by the oral route. Mice receiving mAb 17.41 shed significantly fewer (P < 0.005) C. parvum oocysts than scid mice receiving isotype control mAb. Intestinal (but not gastric) infectivity scores were also reduced for scid mice treated with mAb 17.41 (P < 0.01).

The treatment of intraperitoneal malignant disease with monoclonal antibody guided 131I radiotherapy.

Seven patients with small volume ovarian carcinoma, remaining after conventional therapy with surgery and a platinum containing chemotherapy regimen, were treated with intraperitoneal monoclonal antibody guided radiotherapy. 100 mCi131I conjugated to 10 mg of monoclonal antibody were injected i.p. in 2,000 ml peritoneal dialysis fluid. Patients were evaluated 3 months later; 3 had clinical progressive disease while third look laparotomy demonstrated progressive disease in 3 of the remaining 4 patients. The seventh patient did not have a third look laparotomy and is currently inevaluable for response. Five patients with recurrent malignant ascites not controlled by diuretics or repeated paracentesis were similarly treated with 75-170 mCi131I conjugated to 10 mg monoclonal antibody. In three patients the ascites was controlled for a mean of 4 months. One patient died too early to assess the control of his ascites but tumour cells disappeared from the ascitic fluid after therapy. In the patient whose ascites were not controlled, a subpopulation of antigen-negative tumour cells was demonstrated. This study was unable to demonstrate a therapeutic benefit for i.p. injected monoclonal antibody guided radiotherapy for solid intraperitoneal tumour but suggests that it may be capable of controlling the accumulation of antigen positive malignant ascites.

In vivo depletion of Lyt-2 cells fails to alter acute and relapsing EAE

The cellular mechanisms of recovery and relapses in experimental allergic encephalomyelitis (EAE) are not known. In order to determine the role of the suppressor/cytotoxic T lymphocytes (Lyt-2 +) in EAE we studied the effect of in vivo depletion of this subset using monoclonal antibodies. Intraperitoneal injection of 1 mg of monoclonal antibody 2.43 resulted in rapid depletion of Lyt-2 + cells from lymph node, spleen and blood. Depletion of this subset had no effect on the kinetics of development, severity, and duration of acute EAE. Furthermore, following recovery from acute EAE administration of 2.43 did not result in development of relapses that were different in onset or severity from control animals. These results suggest that T cells of the Lyt-2 phenotype do not play a significant role in the immunoregulation of EAE.

Rat (and mouse) monoclonal antibodies V.A. simple automated technique of antigen purification by immunoaffinity chromatography

A technique to automate the purification of antigens by immunoaffinity chromatography is described. It is operated on a time basis and measurements of the optical density of the immunoaffinity column eluate are made at 280 nm. Immunoaffinity columns of Sepharose 4B coupled mouse or rat monoclonal antibody can be used for 150 or more cycles. From 23 to 120 mg of monoclonal antibody were purified per run. There is a potential application of this system to other forms of affinity chromatography.

One-step immunoaffinity purification of active progesterone receptor. Further evidence in favor of the existence of a single steroid binding subunit.

A very high capacity immunoaffinity matrix for the purification of progesterone receptor was prepared by cross-linking a monoclonal antireceptor antibody to protein A-Sepharose through the Fc fragment. The monoclonal antibody was selected for its property of losing affinity for the receptor at pH 10.5, i.e., in conditions where the receptor remains stable for extensive periods of time. This made it possible to elute active receptor form the immunosorbent. From crude rabbit uterine cytosol the steroid-receptor complexes were purified in a single step. A 1-mL column (containing 7 mg of monoclonal antibody) bound 1600 pmol of steroid-receptor complexes of which 79.5% were eluted. The overall yield of purification was 49%. The specific activity of the purified steroid-receptor complexes was 6.71 +/- 0.79 nmol of bound steroid/mg of protein (mean +/- SE of four experiments). The purified receptor consisted of a mixture of 110 000- and 79 000-dalton forms. The latter appeared to be produced by proteolysis of the larger form during purification since immunoblot experiments showed that, at the start of purification, the 110 000-dalton form was present in overwhelming majority (80-95%) in the uterine cytosol and that the 79 000-dalton form only appeared during purification. This conclusion was also supported by the peptide analysis of both forms of receptor: the purified receptor was denatured and labeled with 125I; the 110 000- and 79 000-dalton forms were isolated by gel electrophoresis in denaturing conditions and electroelution and were then submitted to mild or extensive digestions by trypsin, chymotrypsin, and protease V8 from Staphylococcus aureus.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of autoimmune MRL/Ipr mice with monoclonal antibody to Thy-1.2: a single injection has sustained effects on lymphoproliferation and renal disease.

Abstract MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop an autoimmune disease characterized by anti-DNA antibodies, immune-complex glomerulonephritis, and massive proliferation of a distinct population of T cells. The proliferating T cells have the phenotype Thy-1.2+, T200+, Lyt-1+,2-,3-, but Thy-1.2 and Lyt-1 are expressed in abnormally low density. These cells appear to function as helper cells, and neonatal thymectomy prevents both lymphoproliferation and autoimmunity, which suggests that autoimmunity in MRL/lpr mice is secondary to T cell proliferation. We therefore attempted to reduce lymphoproliferation by treating MRL/lpr mice with a single injection of rat monoclonal antibody (MAb) to Thy-1.2 (30-H12, IgG2b). Mice were treated at 8 wk, before the onset of overt disease. We found that MRL/lpr mice were resistant to depletion of circulating T cells (CTC) by anti-Thy-1.2; 0.6 mg of antibody totally depleted CTC from normal mice, but had little or no effect on CTC in MRL/lpr mice. However, treatment with 6 mg of MAb against Thy-1.2 reduced CTC in MRL/lpr mice by over 70%. Moreover, this single treatment markedly reduced the proliferation of CTC over the ensuing 3 mo, despite clearance of the anti-Thy-1.2 from the circulation within 3 wk. Treated mice maintained better renal function than untreated controls, as assessed by levels of blood urea nitrogen (BUN), although anti-DNA antibodies were not significantly reduced. The effect of anti-Thy-1.2 was specific; treatment with rat MAb to the common leukocyte antigen T200 produced only a transient effect on circulating lymphocytes and did not reduce renal disease. The prolonged effects of a single injection of anti-Thy-1.2 suggest that the MAb produces a sustained alteration in immune regulation. The improvement in renal disease is in accord with evidence that autoimmune disease in MRL/lpr mice is T cell dependent. Monoclonal anti-lymphocyte antibodies may be useful in the treatment of autoimmunity.