Doses Of IgG Research Articles

Preclinical Activity of Brentuximab Vedotin (SGN-35) in Primary Effusion Lymphoma (PEL),

Abstract 3728▪▪This icon denotes a clinically relevant abstractPrimary effusion lymphoma (PEL) is a distinct and aggressive subtype of non-Hodgkin lymphoma (NHL) commonly presenting with pleural, peritoneal, or pericardial malignant effusions usually without a contiguous tumor mass. PEL is most commonly diagnosed in HIV-positive patients, accounting for 4% of all NHLs in this population, yet may also develop in immunosuppressed HIV-negative individuals. While Human Herpes Virus 8 (HHV8 or Kaposi’s sarcoma-associated herpesvirus) is directly implicated in the oncogenesis of this lymphoma, most PEL cases are also associated with Epstein-Barr virus and the combination of the two may facilitate transformation. The tumor cells exhibit plasmablastic features and express CD45, CD38, CD138, HHV8 and CD30. PEL is an aggressive tumor characterized by a short median survival of only 6 months with current therapeutic approaches underscoring the urgent need for development of new therapeutics. Brentuximab vedotin (SGN-35) is an antibody-drug conjugate (ADC) comprised of an anti-CD30 monoclonal antibody cAC10 conjugated by a protease-cleavable dipeptide linker to a potent cell killing agent monomethyl auristatin E (MMAE). Following binding to CD30, brentuximab vedotin is rapidly internalized and is transported to lysosomes, where the peptide linker is selectively cleaved allowing binding of the released MMAE to tubulin and leading to cell cycle arrest and apoptosis. Brentuximab vedotin was recently reported to have promising antitumor activity in CD30 expressing tumors, such as Hodgkin and Anaplastic large cell lymphomas. Since PEL tumors are reported to express CD30, we have hypothesized that brentuximab vedotin might be effective in the treatment of this NHL subtype. Initially, we have confirmed by flow cytometry the expression of CD30 on PEL cell lines (UM-PEL 1, UM-PEL 3, BC-1 and BC-3), and by review of immunohistochemistry and flow cytometry results in patients with previous diagnosis of PEL at our institution. To examine in vitro potency of brentuximab vedotin, UM-PEL 1, UM-PEL 3, BC-1 and BC-3 PEL cell lines were treated with brentuximab vedotin at concentration ranging from 0–100 micrograms/ml. Staining with YO-PRO and Propidium Iodide (PI) demonstrated dose dependent cell apoptosis and death in all the cell lines at 72 hours post treatment. In contrast, control IgG conjugated with MMAE failed to induce apoptosis and cell death of PEL cell lines confirming specific brentuximab vedotin cytotoxicity. Furthermore, brentuximab vedotin decreased proliferation of PEL cells at 48 hours leading to a complete proliferation arrest at 72 hours, as measured by MTS assay. These effects were absent after equivalent doses of control IgG conjugated drug treatment. Supportive to this, labeling of cells with PI to detect active DNA content by flow cytometry showed that bretuximab vedotin induced growth arrest in G2/M phase. To further establish the anti-tumor potential of brentuximab vedotin in vivo, we used the direct xenograft UM-PEL 1 model, established in our laboratory (Sarosiek, PNAS 2010), which mimics human PEL tumors. UM-PEL 1 bearing mice were injected intraperitoneally 3 times a week with brentuximab vedotin or control IgG conjugated MMAE for 4 weeks. Brentuximab vedotin treatment markedly prolonged overall survival of UM-PEL-1 bearing mice compared to controls (p Disclosures:No relevant conflicts of interest to declare.

In vivo effects of a human thyroid-stimulating monoclonal autoantibody (M22) and a human thyroid-blocking autoantibody (K1-70).

PurposeTo study in vivo effects of the human monoclonal TSH receptor (TSHR) autoantibodies M22 (stimulating type) and K1-70 (blocking type) on thyroid hormone levels in rats.MethodsSerum levels of total T4, free T4, M22 and K1-70 were measured following intramuscular injection of M22 IgG (2–4 μg/animal), K1-70 IgG (10–200 μg/animal) or both into rats. Thyroid pathology was assessed in M22-injected rats.ResultsSerum levels of total T4 and free T4 increased in a dose-dependent manner following injection of M22 IgG. Thyroid follicular cell hypertrophy was dependent on the dose of M22 IgG. K1-70 IgG caused a dose dependent decrease of total T4 and free T4 levels in rats receiving K1-70 only. The stimulating effects of M22 IgG on T4 levels in rats were completely inhibited by K1-70 IgG.ConclusionM22 is a potent stimulator of thyroid hormone secretion in vivo. In contrast, K1-70 inhibits thyroid hormone secretion in vivo. Furthermore, K1-70 has the ability to inhibit the stimulating activity of M22 in vivo and as such has potential as a new drug to block TSHR stimulation by autoantibodies in Graves’ disease.

124I-huA33 Antibody PET of Colorectal Cancer

Humanized A33 (huA33) is a promising monoclonal antibody that recognizes A33 antigen, which is present in more than 95% of colorectal cancers and in normal bowel. In this study, we took advantage of quantitative PET to evaluate (124)I huA33 targeting, biodistribution, and safety in patients with colorectal cancer. We also determined the biodistribution of (124)I-huA33 when a large dose of human intravenous IgG (IVIG) was administered to manipulate the Fc receptor or when (124)I-huA33 was given via hepatic arterial infusion (HAI). We studied 25 patients with primary or metastatic colorectal cancer; 19 patients had surgical exploration or resection. Patients received a median of 343 MBq (44.4-396 MBq) and 10 mg of (124)I-huA33. Nineteen patients received the antibody intravenously and 6 patients via HAI, and 5 patients also received IVIG. Ten of 12 primary tumors were visualized in 11 patients. The median concentration in primary colon tumors was 0.016% injected dose per gram, compared with 0.004% in normal colon. The PET-based median ratio of hepatic tumor uptake to normal-liver uptake was 3.9 (range, 1.8-22.2). Quantitation using PET, compared with well counting of serum and tissue, showed little difference. Prominent uptake in bowel hindered tumor identification in some patients. Pharmacokinetics showed that patients receiving IVIG had a significantly shorter serum half-time (41.6 ± 14.0 h) than those without (65.2 ± 9.8 h). There were no differences in clearance rates among the intravenous group, IVIG group, and HAI group, nor was there any difference in serum area under the curve, maximum serum concentration, or volume of distribution. Weak titers of human-antihuman antibodies were observed in 6 of 25 patients. No acute side effects or significant toxicities were associated with huA33. Good localization of (124)I-huA33 in colorectal cancer with no significant toxicity has been observed. PET-derived (124)I concentrations agreed well with those obtained by well counting of surgically resected tissue and blood, confirming the quantitative accuracy of (124)I-huA33 PET. The HAI route had no advantage over the intravenous route. No clinically significant changes in blood clearance were induced by IVIG.

Cryopreserved Ex-Vivo Expanded Murine Hematopoietic Progenitors Can Engraft Across MHC Barriers and Improve Survival In a Murine Model of Acute Radiation Syndrome.

AbstractAbstract 1463 Introduction:Practical clinical countermeasures that could enhance survival after accidental or deliberate radiation exposure are lacking. We have previously demonstrated that culture of murine hematopoietic stem/progenitor cells in the presence of immobilized Notch ligand Delta1 results in a multi-log increase in the number of lin-/sca-1+/c-kit+ (LSK) cells with short term lymphoid and myeloid repopulating ability. Here we show that Delta1 cultured LSK cells, cryopreserved after expansion, can be an effective therapy in a murine model of the hematopoietic acute radiation syndrome (hARS). Methods:Whole bone marrow (BM) was obtained from Ly5a mice (H-2b, CD45.1). LSK cells were isolated by flow cytometry and placed in culture in the presence of either immobilized Delta1 or control human IgG. Serum free conditions were used, consisting of Iscove's medium supplemented with cytokines mSCF, hFlt-3 ligand, hIL-6, and hIL-11. After 14 days, expanded cells were harvested and cryopreserved in 90% FBS + 10% DMSO. On the day of transplant, frozen cells were thawed, washed, and resuspended in PBS + 1% FBS; manual viable cell counts and LSK phenotyping were performed prior to tail vein infusion with escalating doses of Delta1 or IgG cultured cells at doses of 1, 3, 5, or 10 × 106 (Delta-1 group only at 10×106) into supralethally irradiated (8.5 Gy) MHC-mismatched Balb/c mice (H-2d, CD45.2). Peripheral blood (PB) and BM were collected from mice at 1, 2, 3 and 4 weeks after transplantation for chimerism determination by cytofluorometry. Results:Culture with Delta1 resulted in significantly greater increases in absolute numbers of LSK cells (7.2 × 104-fold expansion) as compared to growth with control IgG (0.8 × 104-fold expansion). Approximately 90% of viable LSK cells were recovered post thaw. PB samples from recipient Balb/c mice receiving Delta1 cells demonstrated significantly higher Ly5a+ donor cell engraftment as compared to recipients receiving IgG-cultured LSK cells (p=0.0001). Donor IgG cultured cells were detectable only at day 7, whereas cells grown in the presence of Delta1 persisted through day 14 and 21. Only mice transplanted with Delta1-expanded cells showed engraftment in marrow, although by 2 weeks donor cells had decreased substantially. No signs of graft versus host disease (GVHD) were observed. Survival was significantly prolonged among mice that received Delta1-cultured cells, whereas all mice that received IgG cultured cells died within the first 3 weeks after irradiation (p=0.0001). Overall survival at day 30 was 11, 20, 26 and 63 percent after receiving 1, 3, 5 and 10 × 106 Delta1-cultured cells, respectively. Mice that received 10 × 106 cultured cells showed a statistically significant better survival (p=0.02), demonstrating a dose response relationship with the highest survival observed in mice that received the highest dose of expanded cells. Conclusions:Using the Notch ligand Delta1 for the ex vivo expansion of murine hematopoietic progenitor cells, we have demonstrated that the cultured cells can be efficiently cryopreserved without loss of in vivo function. Infusion of Notch-expanded and cryopreserved cells into lethally irradiated mismatched recipients demonstrated that short-term engraftment without manifestations of GVHD can be achieved across major H-2 barriers and resulted in. significantly enhanced survival in a dose dependent manner. We have previously demonstrated that culture of human cord blood CD34+ cells in the presence of Delta1 also results in a significant increase in the absolute number of hematopoietic progenitor cells that are capable of rapid myeloid reconstitution in vivo. The findings presented herein thus support further development of a parallel human ex vivo expanded and cryopreserved product for clinical application in a non-HLA matched setting. Disclosures:No relevant conflicts of interest to declare.

Anti-PrPC monoclonal antibody infusion as a novel treatment for cognitive deficits in an Alzheimer's disease model mouse.

BackgroundAlzheimer's Disease (AD) is the most common of the conformational neurodegenerative disorders characterized by the conversion of a normal biological protein into a β-sheet-rich pathological isoform. In AD the normal soluble Aβ (sAβ) forms oligomers and fibrils which assemble into neuritic plaques. The most toxic form of Aβ is thought to be oligomeric. A recent study reveals the cellular prion protein, PrPC, to be a receptor for Aβ oligomers. Aβ oligomers suppress LTP signal in murine hippocampal slices but activity remains when pretreated with the PrP monoclonal anti-PrP antibody, 6D11. We hypothesized that targeting of PrPC to prevent Aβ oligomer-related cognitive deficits is a potentially novel therapeutic approach. APP/PS1 transgenic mice aged 8 months were intraperitoneally (i.p.) injected with 1 mg 6D11 for 5 days/week for 2 weeks. Two wild-type control groups were given either the same 6D11 injections or vehicle solution. Additional groups of APP/PS1 transgenic mice were given either i.p. injections of vehicle solution or the same dose of mouse IgG over the same period. The mice were then subjected to cognitive behavioral testing using a radial arm maze, over a period of 10 days. At the conclusion of behavioral testing, animals were sacrificed and brain tissue was analyzed biochemically or immunohistochemically for the levels of amyloid plaques, PrPC, synaptophysin, Aβ40/42 and Aβ oligomers.ResultsBehavioral testing showed a marked decrease in errors in 6D11 treated APP/PS1 Tg mice compared with the non-6D11 treated Tg groups (p < 0.0001). 6D11 treated APP/PS1 Tg mice behaved the same as wild-type controls indicating a recovery in cognitive learning, even after this short term 6D11 treatment. Brain tissue analysis from both treated and vehicle treated APP/PS1 groups indicate no significant differences in amyloid plaque burden, Aβ40/42, PrPC or Aβ oligomer levels. 6D11 treated APP/PS1 Tg mice had significantly greater synaptophysin immunoreactivity in the dentate gyrus molecular layer of the hippocampus compared to vehicle treated APP/PS1 Tg mice (p < 0.05).ConclusionsEven short term treatment with monoclonal antibodies such as 6D11 or other compounds which block the binding of Aβ oligomers to PrPC can be used to treat cognitive deficits in aged AD transgenic mice.

Abstract 5543: Combining antibody-targeted radiation (radioimmunotherapy) and antibody-SN-38 conjugates (ADC) improves pancreatic cancer therapy

Abstract We previously reported effective anti-tumor activity in nude mice bearing human pancreatic tumors with 90Y-humanized PAM4 IgG (hPAM4; 90Y-clivatuzumab tetrexetan) that was enhanced when combined with gemcitabine (GEM). These studies led to clinical testing of fractionated 90Y-hPAM4 IgG combined with GEM that is showing encouraging objective responses. While GEM is known for its radiosensitizing ability, alone it is not a very effective therapeutic agent for pancreatic cancer and its dose is limited by hematologic toxicity, which is also limiting for 90Y-hPAM4 IgG. We recently reported promising anti-tumor activity with an ADC composed of hRS7 IgG (humanized anti-epithelial glycoprotein-1; EGP-1) and SN-38, the active component of irinotecan. This ADC is very well tolerated in mice (eg, ≥ 60 mg), yet just 4.0 mg (0.5 mg, twice-weekly x 4) is significantly therapeutic. Since EGP-1 is also expressed in most pancreatic cancers, we have examined combinations of 90Y-hPAM4 IgG with RS7-SN-38 in nude mice bearing 0.35 cm3 subcutaneous xenografts of the human pancreatic cancer cell line, Capan-1. Mice (n=10) were treated with a single dose of 90Y-hPAM4 IgG alone (130 µCi, ie, the maximum tolerated dose or 75 µCi), with RS7-SN-38 alone (as above), or combinations of the 2 agents at the two 90Y-hPAM4 dose levels, with the first ADC injection given the same day as the 90Y-hPAM4. All treatments were tolerated, with ≤ 15% loss in body weight. Objective responses occurred in most animals, but they were more robust in both of the combination groups as compared to each agent given alone. All animals in the 0.13 mCi 90Y-hPAM4 IgG + hRS7-SN-38 achieved a tumor-free state within 4 weeks, while other animals continued to have evidence of persistent disease. These studies provide the first evidence that combined radioimmunotherapy and ADC can enhance efficacy at safe doses. (Supported in part by SBIR grant 2R44CA1148 from the NCI.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5543.

Improving the treatment of non‐Hodgkin lymphoma with antibody‐targeted radionuclides

Radioimmunotherapy of non-Hodgkin lymphoma comprises a (90)Y- or (131)I-labeled murine anti-CD20 IgG, but both agents also include a substantial dose of unlabeled anti-CD20 IgG given immediately before the radioconjugate to reduce its uptake in the spleen (primary normal B-cell antigen sink); this extends its plasma half-life and improves tumor visualization. Thus, these treatments combine an effective anti-CD20 radioconjugate with an unconjugated anti-CD20 antibody that is also therapeutically active, but the large anti-CD20 IgG predose ( approximately 900 mg) may diminish the tumor localization of the radioimmunoconjugate (eg, 10-35 mg). We have examined alternative approaches that enhance radionuclide targeting and improve antitumor responses. One uses a (90)Y-labeled anti-CD22 IgG (epratuzumab) combined with an antibody therapy regimen of a humanized anti-CD20 IgG (veltuzumab). Pretargeted radionuclide therapy using a trivalent, humanized, recombinant bispecific anti-CD20 antibody with a (90)Y-hapten-peptide is another highly effective method that is also less toxic than directly radiolabeled IgG. Finally, all approaches benefit from the addition of a consolidation-dosing regimen of the anti-CD20 IgG antibody. This article reviews these various options and discusses how some fundamental changes could potentially enhance the response and duration from radionuclide-targeted therapy.

Transient Anti-CD40L Co-Stimulation Blockade Prevents Immune Responses against Human Bullous Pemphigoid Antigen 2: Implications for Gene Therapy

Skin grafts from mice expressing human bullous pemphigoid antigen 2 (hBPAG2) in epidermal basement membrane elicit hBPAG2-specific IgG and graft loss in wild-type (Wt) recipients. Graft loss was dependent on CD4+ T cells and correlated with the production and tissue deposition of hBPAG2-specific IgG. To explore the role of CD40/CD40 ligand (CD40L) interaction in this model, Wt mice grafted with transgenic (Tg) skin were treated with hamster anti-CD40L mAb MR1. In contrast to grafted Wt mice treated with equivalent doses of control IgG, 22 of 23 MR1-treated Wt mice did not develop hBPAG2-specific IgG or graft loss for >or=60 days. MR1-treated mice also accepted a second Tg skin graft without durable production of hBPAG2-specific IgG or graft loss. Moreover, splenocytes and enriched CD4+ T cells from MR1-treated graft recipients transferred un- or hyporesponsiveness to hBPAG2 to other mice and demonstrated a dominant tolerant effect over cotransferred naive splenocytes following adoptive transfer to Rag2-/- mice. Successful inhibition of hBPAG2-specific IgG production and Tg graft loss following CD40:CD40L co-stimulatory blockade in this model provides opportunities to study mechanisms of peripheral tolerance and generate antigen-specific regulatory CD4+ cells-issues of relevance to patients with pemphigoid as well as individuals undergoing gene replacement therapy for epidermolyis bullosa.

The Effect of a Chimeric Human/Murine Anti-Cocaine Monoclonal Antibody on Cocaine Self-Administration in Rats

The predominantly human sequence anti-cocaine monoclonal antibody (mAb), 2E2, has high affinity and specificity for cocaine and antagonizes cocaine distribution to the brain in mice. To determine whether 2E2 can alter the self-administration of cocaine in rats, both cocaine-induced reinstatement (priming) of self-administration, and the rates of cocaine consumption were assessed during daily sessions. After self-administration training, the rats' cocaine priming threshold values were stable over a 2-week baseline period. Furthermore, the rates of cocaine consumption at unit doses of 0.3 and 3.0 micromol/kg were steady within sessions and stable between sessions. Then, 2E2 (120 mg/kg i.v.) or an equivalent dose of nonspecific human polyclonal IgG (control) was infused and daily sessions continued. 2E2 produced an initial, approximately 3-fold, increase in the cocaine priming threshold that declined toward baseline values over the subsequent 3 weeks, with an effect t((1/2)) of approximately 4 days. In contrast to the substantial increase in the cocaine priming threshold, 2E2 produced only modest dose-dependent increases (42 and 18%) in the cocaine consumption rates, and these also gradually declined toward baseline values. There was no significant effect of the control IgG on the priming threshold or rates of consumption of cocaine. After infusion, antibody blood concentrations declined over time, and a two-compartment pharmacokinetic model generated values for the distribution and elimination half-lives of 0.5 and 11.6 days for 2E2 and 0.4 and 6.0 days for control IgG. 2E2 had a long-lasting effect on cocaine-induced priming, which may predict its efficacy as an immunotherapy for cocaine abuse.

Recognition, Clinical Diagnosis and Management of Patients With Primary Antibody Deficiencies: A Systematic Review

Wood P, Stanworth S, Burton J, et al. Clin Exp Immunol. 2007;149(3):410–423 PURPOSE OF THE STUDY. To create an evidence-based literature review of clinical diagnosis and management of primary antibody deficiency. METHODS. Computer literature searches were conducted for randomized clinical trials in medical literature databases including the US National Library of Medicine (Medline), the Excerpta Medica database (EMBASE), the Cochrane Library, the Database of Abstracts of Reviews of Effects (DARE) of the Centre for Reviews and Dissemination (University of York), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL) up to June 2006. Reports were rated on the basis of relevance and quality or type of evidence. Disease entities included were X-linked agammaglobulinemia, common variable immunodeficiency, hyper–immunoglobulin M (IgM) syndromes, IgG subclass deficiency with and without IgA deficiency, and specific antibody deficiency. Reports that involved only a limited number of patients were not included. RESULTS. Individuals who present with recurrent respiratory infections of all types, especially with excessive frequency and severity, should be screened for antibody deficiency. In children, common associations included growth delay and failure to thrive, recurrent fevers without a source, and poor school attendance or performance. Chronic diarrhea was found in 40% to 60% of the patients at all ages. The median delay in diagnosis was 1 year but ranged to &amp;gt;10 years. Delayed diagnosis led to increased risk of bronchiectasis, pulmonary hypertension, and cor pulmonale. Randomized trials of the efficacy of γ-globulin replacement versus placebo do not currently exist. Many observational studies have confirmed the benefit of IgG therapy for reducing infectious morbidity. Higher IgG doses are associated with reduced incidence and severity of infections. CONCLUSIONS. Delayed diagnosis was common as a result of lack of recognition of presenting symptoms and signs. Delay was less frequent when patients were referred to specialists. Delayed diagnosis led to delayed therapy (IgG) and a higher rate of infections and morbidity. Several areas for additional research were identified, including studies of efficacy and dose of IgG and adjunct therapies (antibiotics), microbiologic study of pathogens, identification of prognostic markers, and effective monitoring of disease (eg, lung function, other organs, cancer). Collaboration and pooling of data among centers nationally and internationally would likely lead to better data. REVIEWER COMMENTS. This study again points out the human cost of delayed or undiagnosed immunodeficiency and the difficulties in accumulating high-quality data on therapy and outcomes. Several initiatives are taking shape in the United States and abroad to collaborate to answer many of the remaining questions pointed out in this study.

Response to “Inadequate prophylaxis in anti-D immunisation”.

Dear Sirs, the comments made by Professor Reali1 on the Case Report, “Anti-D immunisation with permanent biological damage following inadequate post-amniocentesis prophylaxis2” are all pertinent and I thank him for the attention he gave to the report and for the occasion to make some precisions on the case presented, which was an example of failed prophylaxis with anti-D IgG, since it is now known that amniocentesis can act as a stimulus to maternal immunisation because of the possibility of repeated transplacental haemorrhages. Before submitting the case we considered other possible causes of the failure of the prophylaxis and, on the basis of the official documentation and the information provided directly by the patient, we were able to be sure that the prophylaxis was truly administered and at the right time. The initial serological examination was carried out twice with the same outcome (negative), the method used in the hospital which referred the patient is the same as that used in our centre (microcolumn agglutination test) and we have no reasons to doubt the correct performance of the test. It seems impossible that the dose of anti-D IgG was different from that declared and recorded, since there are no other doses of anti-D IgG in the referring hospital. For all the above reasons and because of the outcome, we felt that it was useful to report this as a case of failed prophylaxis attributable to a discrepancy between the antigen load in the mother and the amount of antibodies injected. It is, in fact, agreed that the dose of the treatment should be commensurate with the entity of the foetal-maternal haemorrhage3, and it is known that inadequate prophylaxis is the most frequent cause of failure of treatment. We, therefore, felt that it was worth drawing attention to the need to carry out a quantitative evaluation of foetal-maternal haemorrhage also in cases of amniocentesis and/or trauma during pregnancy, just as we feel that it is safer to adapt the therapy to the amount of haemorrhage, rather than injecting a single, arbitrarily chosen dose, because of the numerous variables that can have effects and because of the severity of the consequences of immunisation, which can lead, as in our case, to permanent biological damage.

Critical role of peripheral blood phagocytes and the involvement of complement in tumour necrosis factor enhancement of passive collagen-arthritis.

Studies have implicated tumour necrosis factor-alpha (TNF-alpha) in type-II collagen (CII)-induced arthritis (CIA), a well established animal model of human rheumatoid arthritis. Precisely how TNF is involved in CIA is not yet clear. In this study the effects of TNF on CIA were examined, independent of its potential effects on the immune response, by performing peri-articular injection of TNF in combination with passive immunization of rats. A sub-arthritic dose (5 mg) of affinity-purified anti-CII IgG, which alone was insufficient to induce spontaneous clinical arthritis, was used throughout the study. Obvious clinical arthritis that persisted for several days was rapidly induced by injections of 100 ng TNF into hindpaws of rats that were passively immunized shortly before the TNF injection. Injections of TNF in non-immunized control rats did not induce clinical arthritis, nor did buffer-only injections in passively immunized controls. The clinical arthritic response was a local phenomenon, limited only to the TNF-injected hindpaws. No swelling was observed in the opposite, buffer-injected hindpaws, indicating the effects of TNF were not systemic. Depletion of peripheral blood phagocytes with anti-rat neutrophil antiserum before passive immunization completely abolished the ability of TNF to induce clinical arthritis, identifying phagocytic cells as the essential target cells in evoking this arthritic response. A role for complement activation was also demonstrated in this model through the use of a soluble recombinant version of CD35, the cell surface complement receptor type-1 (sCR1, BRL55730), which significantly reduced TNF-induced arthritis in phagocyte-replete rats.

Increased levels of soluble low-affinity Fc gamma receptors (IgG-binding factors) in the sera of tumour-bearing mice.

Soluble forms of low affinity Fc gamma receptors (Fc gamma R), also called IgG-binding factors (IgG-BF), have been shown to play a regulatory role in immune responses. By using an immunodot assay with the anti-mouse Fc gamma R MoAb, 2.4G2, the levels of IgG-BF have been measured in the sera of mice bearing syngeneic tumours of lymphoid or non-lymphoid origin or in mice injected with high doses of murine IgG. These sera contained large amounts of IgG-BF as compared with controls. In the case of mice bearing IgG2a- or IgG2b-secreting hybridomas or lymphomas, serum IgG-BF increased progressively with tumour size and serum monoclonal IgG concentration, reaching 4-12 times the normal levels. A less than three-fold increase was found in mice bearing an IgG1-secreting hybridoma or tumours which do not secrete IgG (IgA-secreting hybridoma, non-immunoglobulin-secreting lymphoid tumours or melanoma) or in mice injected with 9 mg of monoclonal IgG2a. The enhancement of serum IgG-BF levels was independent of the expression of Fc gamma R by the tumour cells, suggesting that the majority of IgG-BF secreted in response to tumours was produced by the host rather than by the tumour. The increased production of IgG-BF may participate in the control of tumour growth and in the modulation of the host immune responses in tumour-bearing animals.

Prominent Plasmacytosis Following Intravenous Immunoglobulin Correlates with Clinical Improvement in Guillain-Barré Syndrome

BackgroundHigh doses of pooled polyclonal IgG are commonly used to treat numerous autoimmune diseases. Their mode of action nevertheless remains only partially explained. At the same time, until now, no early biological marker has been able to predict their efficacy.Methodology/Principal FindingsIn a first pilot retrospective analysis, we reviewed white blood cell counts and blood smears in consecutive patients with autoimmune disease (n = 202) and non-autoimmune disease (n = 104). Autoimmune patients received either intravenous immunoglobulin (IVIg, n = 103), plasma exchange (n = 78) or no specific treatment (n = 21). We then prospectively monitored consecutive autoimmune patients with IVIg injection (n = 67), or without any specific treatment (n = 10) using the same routine laboratory tests, as well as flow cytometry. Both retrospective and prospective analyses identified large plasma-cell mobilization exclusively in IVIg-treated autoimmune patients 7 days after initiation of treatment. The majority of IVIg-mobilized plasma cells were immature HLA-DRhigh/CD138low/CXCR4low plasma cells expressing intracellular immunoglobulin G which were neither IVIg- nor human IgG-specific. Importantly, we found a strong negative correlation between the absolute number of IVIg-mobilized plasma cells and time to improve neurological function in both retrospective and prospective studies of Guillain-Barré syndrome (GBS), (r = −0.52, p = 0.0031, n = 30, r = −0.47, p = 0.0028, n = 40, respectively).Conclusions/SignificanceIVIg promotes immature plasma-cell mobilization in patients with GBS, chronic inflammatory demyelinating polyneuropathy, myasthenia gravis and inflammatory myopathy. Prominent day 7 plasma-cell mobilization is a favourable prognostic marker in patients with GBS receiving IVIg treatment.

A human anti‐cocaine monoclonal antibody antagonizes the cocaine‐induced reinstatement of self‐administration in rats

Immunotherapy may represent a viable alternative to pharmacotherapy for cocaine abuse. The predominantly human sequence anti‐cocaine monoclonal antibody, 2E2, has high affinity (Kd = 4 nM) and specificity for cocaine. In mice, 2E2 sequesters cocaine in the peripheral circulation, thereby decreasing brain cocaine concentrations. Therefore, it was hypothesized that 2E2 would antagonize cocaine‐induced reinstatement (priming) of self‐administration, a partial model of relapse that depends on the brain cocaine concentration. The level of cocaine required to reinstate self‐administration (priming threshold) in trained rats was calculated during daily sessions run 6 days a week. After 2 weeks of baseline measurements, rats were infused with 2E2 (120 mg/kg i.v.) or an equivalent dose of human polyclonal IgG as a control and daily sessions resumed. 2E2 produced a significant almost 3‐fold increase in priming threshold that gradually declined towards baseline values. The control IgG had no effect. The terminal elimination half‐life of 2E2 was 10 days. 2E2 also produced an approximately 20–40% increase in the rate of maintained cocaine self‐administration, which declined over days. This is less than the increase typically produced by competitive dopamine receptor antagonists. 2E2 may represent a therapeutic agent for decreasing the probability of relapse in cocaine abusers. Supported by PHS grant DA018538.

Rapid generation of an anthrax immunotherapeutic from goats using a novel non-toxic muramyl dipeptide adjuvant

BackgroundThere is a clear need for vaccines and therapeutics for potential biological weapons of mass destruction and emerging diseases. Anthrax, caused by the bacterium Bacillus anthracis, has been used as both a biological warfare agent and bioterrorist weapon previously. Although antibiotic therapy is effective in the early stages of anthrax infection, it does not have any effect once exposed individuals become symptomatic due to B. anthracis exotoxin accumulation. The bipartite exotoxins are the major contributing factors to the morbidity and mortality observed in acute anthrax infections.MethodsUsing recombinant B. anthracis protective antigen (PA83), covalently coupled to a novel non-toxic muramyl dipeptide (NT-MDP) derivative we hyper-immunized goats three times over the course of 14 weeks. Goats were plasmapheresed and the IgG fraction (not affinity purified) and F(ab')2 derivatives were characterized in vitro and in vivo for protection against lethal toxin mediated intoxication.ResultsAnti-PA83 IgG conferred 100% protection at 7.5 μg in a cell toxin neutralization assay. Mice exposed to 5 LD50 of Bacillus anthracis Ames spores by intranares inoculation demonstrated 60% survival 14 d post-infection when administered a single bolus dose (32 mg/kg body weight) of anti-PA83 IgG at 24 h post spore challenge. Anti-PA83 F(ab')2 fragments retained similar neutralization and protection levels both in vitro and in vivo.ConclusionThe protection afforded by these GMP-grade caprine immunotherapeutics post-exposure in the pilot murine model suggests they could be used effectively to treat post-exposure, symptomatic human anthrax patients following a bioterrorism event. These results also indicate that recombinant PA83 coupled to NT-MDP is a potent inducer of neutralizing antibodies and suggest it would be a promising vaccine candidate for anthrax. The ease of production, ease of covalent attachment, and immunostimulatory activity of the NT-MDP indicate it would be a superior adjuvant to alum or other traditional adjuvants in vaccine formulations.

The antiinflammatory activity of IgG: the intravenous IgG paradox

How high doses of intravenous IgG (IVIG) suppress autoimmune diseases remains unresolved. We have recently shown that the antiinflammatory activity of IVIG can be attributed to a minor species of IgGs that is modified with terminal sialic acids on their Fc-linked glycans. Here we propose that these Fc-sialylated IgGs engage a unique receptor on macrophages that, in turn, leads to the upregulation of an inhibitory Fcγ receptor (FcγR), thereby protecting against autoantibody-mediated pathology.

A survey of the current use of anti-D immunoprophylaxis and the incidence of haemolytic disease of the newborn in Italy.

The Italian Society of Transfusion Medicine and Immunohaematology (SIMTI) carried out a survey on the current use of anti-D immunoprophylaxis (IP) in Italy, on its ways of use and on the impact that it has had on decreasing haemolytic disease of the newborn (HDN), due to maternal-foetal incompatibility for the D antigen. The survey was carried out using a questionnaire prepared by the Working Group established for this purpose by the SIMTI. The questions were divided into five groups: the ways of carrying out IP, evaluation of foetal-maternal haemorrhage, serological tests after IP, the current incidence of HDN, and data on exchange transfusions. Data were obtained from 69 Transfusion Services (TS). Four of these give IP antenatally, whereas in the remaining cases IP is given after birth. Almost all the TS evaluate the amount of foetal-maternal haemorrhage in order to give additional doses of anti-D IgG, with the most widely used method being the Kleihauer-Betke test. Data were collected from 176,010 pregnancies: 18,639 were D-negative women, of whom 18,440 were not immunised. There were 136 cases of HDN with anti-D antibodies, and 39 of these required exchange transfusions (ET). Furthermore, there were 1,535 pregnant women with anti-A and/or anti-B IgG, which were clinically significant in 83 and required ET in 37. Finally, 40 women had antibodies, directly related to the pregnancy, against antigens other than D (in eight of these cases ET was necessary). The survey carried out by SIMTI was able to give a sufficiently full and accurate picture of current Italian practices concerning the use and ways of use of anti-D IP in pregnancy and the puerperum, as well as the incidence and characteristics of HDN. Furthermore, this survey was the basis for guidelines on the management of HDN, produced by SIMTI in collaboration with the Italian Society of Obstetricians and Gynaecologists.

Oral immunization of mice with plant-derived fimbrial adhesin FaeG induces systemic and mucosal K88ad enterotoxigenicEscherichia coli-specific immune responses

The importance of adhesins in pathogenicity has resulted in them being useful targets in the defense against bacterial infections. To produce edible vaccines against piglet diarrhea caused by enterotoxigenic Escherichia coli (ETEC), plants were genetically engineered to produce recombinant fimbrial adhesin FaeG. To evaluate the efficacy of the edible vaccine FaeG in mice, the soluble protein extracts were examined by about 15 microg recombinant FaeG for each oral immunization dose per mouse. After four doses of vaccination, both IgG and IgA antibodies specific to K88ad fimbriae were elicited in serum, and specific IgA antibodies were also evoked in feces of the immunized mice. Moreover, visible K88ad ETEC agglutination by the specific serum from the immunized mice was observed, implying the antibody was highly specific and effective. Results from an in vitro villous-adhesion assay further confirmed that serum antibodies of the immunized mice could inhibit K88ad ETEC from adhering to pig intestinal receptors, further demonstrating the oral immune efficacy of the plant-derived FaeG. This study provides a promising, noninvasive method for vaccinating swine by feeding supplements of transgenic plant. Moreover, the low cost and ease of delivery of this edible ETEC vaccine will facilitate its application in economically disadvantaged regions.

Complement-Mediated Mechanisms in Anti-GD2 Monoclonal Antibody Therapy of Murine Metastatic Cancer

The role of complement in antibody therapy of cancer is in general poorly understood. We used the EL4 syngeneic mouse model of metastatic lymphoma to investigate the role of complement in immunotherapy directed against GD2, a target of clinical relevance. IgG2a and IgM anti-GD2 therapy protected EL4-challenged mice from metastases and prolonged survival. Expression of CD59, an inhibitor of direct complement-mediated cytotoxicity (CMC), effectively protected EL4 cells from CMC in vitro but did not affect the outcome of monoclonal antibody therapy. Protection by IgG therapy was also unaffected in mice deficient in C3 or complement receptor 3 (CR3) but was almost completely abrogated in FcgammaR I/III-deficient mice. These data indicate a crucial role for antibody-dependent cell-mediated cytoxicity (ADCC). However, at lower doses of IgG, therapeutic effect was partially abrogated in C3-deficient mice, indicating complement-mediated enhancement of ADCC at limiting IgG concentration. In contrast to IgG, the therapeutic effect of IgM was completely abrogated in C3-deficient mice. High level expression of CD59 on EL4 did not influence IgM therapy, suggesting IgM functions by complement-dependent cell-mediated cytotoxicity (CDCC), a mechanism thought to be inactive against tumor cells. Thus, IgG and IgM can operate via different primary mechanisms of action, and CDCC and complement-dependent enhancement of ADCC mechanisms are operative in vivo. The effects of complement can be supplemental to other antibody-mediated mechanisms and likely have increased significance at limiting antibody concentration or low antigen density.