Suppression Of Antibody Formation Research Articles

Post-trial follow-up after a randomized clinical trial of COVID-19 convalescent plasma

Background: COVID-19 convalescent plasma (CP) proved to be a safe acute intervention, however, the long-term clinical effects of COVID-19 CP are to date unknown. CP might have a prospective negative effect by down-regulating the inflammatory response suppressing antibody formation and promoting autoantibodies against interferons. Our objective was to establish the long-term safety profile of COVID-19 CP and determine if its administration increases the risk for further respiratory infections in older adults. Methods: All participants included in the intention to treat analysis of a randomized clinical trial evaluating the efficacy of COVID-19 CP in older adults were invited to participate in this post-trial follow-up study. Patients were strictly followed for at least 6 months after randomization. The primary endpoint was the number of patients with clinically confirmed acute respiratory infections (ARIs). Secondary endpoints included all-cause mortality, time to first respiratory infection, SARS-CoV-2 re-infection, adverse events, and persistence of COVID-19 symptoms after initial infection. Results: 142 patients were included in the study (total retention rate=92.8%). The mean age was 77.2 years (SD=8.6) and the median duration of follow-up was 10.4 months (IQR=1.63), with no differences among groups. 20 patients had a clinically confirmed ARI during the study. No differences were observed between groups in the proportion of ARIs (CP=11/72 and Placebo=9/70, p-value=0.678) and in the probability of ARI-free survival between groups (log-rank test p-value=0.63). No differences emerged when comparing groups regarding secondary endpoints. Conclusions: COVID-19 convalescent plasma remains a safe intervention without increasing the risk of acute respiratory infection or other clinical consequences in the long term.

Lettuce Plants Expressing High Levels of Factor VIII Antigen in the Chloroplast for Oral Tolerance in Hemophilia a

Inhibitor formation is a serious complication in factor VIII (FVIII) replacement therapy for the X-linked bleeding disorder hemophilia A and occurs in 20-30% of patients. No prophylactic tolerance protocols exist. In previous proof-of-principle work, we demonstrated oral tolerance induction to FVIII in hemophilia A mice using frozen tobacco leaf cells expressing the heavy chain (HC) and C2 domain of human B domain-deleted (BDD) FVIII (Blood 124:1659-68, 2014). This approach is based on expression of heterologous proteins in the chloroplasts of green leaves and natural bioencapsulation provided by plant cell wall, so that the antigen is protected until its release in the small intestine. The antigens are expressed as fusions with the transmucosal carrier CTB proteins for effective targeting of the gut epithelium and translocation to the immune system. However, significant hurdles toward clinical feasibility remained that we now successfully addressed. Until now, methods to express large human proteins in chloroplasts of edible crop plants did not exist. Building on our recently established gene transfer technology for lettuce chloroplasts and identification of powerful posttranscriptional regulatory elements, we focused on the development of chloroplast genomics tools (e.g. bioinformatics tools for codon optimization and assays that identify sites of ribosome stalling), which ultimately allowed us to express high levels of FVIII antigen in edible crop plants that are suitable for clinical application. Remarkably, codon-optimization improved expression of FVIII heavy chain (HC) and light chain (LC) by15- to 42-fold. Hence, the entire BDD-FVIII sequence can be orally delivered, thereby covering the entire patient population regardless of individual CD4+ T cell epitope usage. A clinical grade manufacturing process is in place, and lyophilization of leaf cells generates material that is stable at ambient temperature for at least 2 years. Testing in hemophilia A mice showed effectiveness of low FVIII antigen doses upon delivery of lyophilized lettuce cells. Repeated oral gavage (twice/week for 2 months) of a mixture of CTB-HC and CTB-C2 suppressed subsequent inhibitor formation against intravenous FVIII (1IU, once/ week for 4 weeks) in hemophilia A mice as well as the original tobacco material, using very low doses of 0.15-0.5 ug/antigen/mouse. When using CTB-HC/CTB-LC combination, a dose of 1.5 ug was most optimal and suppressed inhibitor titers by ~1 log (avg. 4.7 BU/ml vs 44 BU/ml in controls; 50% of tolerized mice had 0-3 BU/ml, while 86% of controls developed 6-126 BU/ml, n=12-14, P <0.05). Translational oral tolerance studies in humans would benefit from cellular biomarkers. Our prior studies showed induction of two subsets of Treg that suppress antibody formation against FVIII, namely CD4+CD25+FoxP3+ Treg and LAP+CD4+CD25-FoxP3- Treg. The latter stain positive on the cell surface for LAP (latency-associated peptide) because of overexpression of TGF-b. Although we found increases in the frequencies of LAP+CD4+CD25-FoxP3- Treg in peripheral blood, a statistical significance was not always observed when compared to the blood of control mice that received intravenous FVIII but not oral tolerance. However, we found significant and consistent increases in the frequencies of circulating LAP-expressing CD4+CD25+FoxP3+ Treg in tolerized mice, suggesting that LAP may be a suitable cellular biomarker for plant-based oral tolerance induction. In conclusion, clinical candidates for oral tolerance to FVIII are now available in form of lettuce plants expressing immunogenic domains or the entire antigen. DisclosuresHerzog:Novo Nordisk: Research Funding; Applied Genetic Technologies Corporation: Consultancy; Spark Therapeutics: Patents & Royalties. Daniell:Novo Nordisk: Research Funding.

CAR- and TRuC-redirected regulatory T cells differ in capacity to control adaptive immunity to FVIII

Regulatory T cells (Tregs) control immune responses in autoimmune disease, transplantation, and enable antigen-specific tolerance induction in protein-replacement therapies. Tregs can exert a broad array of suppressive functions through their T cell receptor (TCR) in a tissue-directed and antigen-specific manner. This capacity can now be harnessed for tolerance induction by “redirecting” polyclonal Tregs to overcome low inherent precursor frequencies and simultaneously augment suppressive functions. With the use of hemophilia A as a model, we sought to engineer antigen-specific Tregs to suppress antibody formation against the soluble therapeutic protein factor (F)VIII in a major histocompatibility complex (MHC)-independent fashion. Surprisingly, high-affinity chimeric antigen receptor (CAR)-Treg engagement induced a robust effector phenotype that was distinct from the activation signature observed for endogenous thymic Tregs, which resulted in the loss of suppressive activity. Targeted mutations in the CD3ζ or CD28 signaling motifs or interleukin (IL)-10 overexpression were not sufficient to restore tolerance. In contrast, complexing TCR-based signaling with single-chain variable fragment (scFv) recognition to generate TCR fusion construct (TRuC)-Tregs delivered controlled antigen-specific signaling via engagement of the entire TCR complex, thereby directing functional suppression of the FVIII-specific antibody response. These data suggest that cellular therapies employing engineered receptor Tregs will require regulation of activation thresholds to maintain optimal suppressive function.

Effects of activation of skin ion channels TRPM8, TRPV1, and TRPA1 on the immune response. Comparison with effects of cold and heat exposure.

The effects of pharmacological stimulation of skin ion channels TRPA1, TRPM8, TRPV1 on the immune response are presented. These effects are compared with the effects of different types of temperature exposures - skin cooling, deep cooling, and deep heating. This analysis allows us to clear the differences in the influence on the immune response of thermosensitive ion channels localized in the skin; (2) whether the changes in the immune response under temperature exposures are due to these thermosensitive ion channels. Experiments were performed on Wistar rats. For stimulation of TRPM8 ion channel, an application to the skin of 1% menthol was used, for TRPA1 - 0.04% allylisotiocianate, and for TRPV1 - capsaicin in a concentration of 0.001.The antigen binding in the spleen was two-times stimulated by activation of the cold-sensitive ion channel TRPM8 and much weaker by activation of warm-sensitive TRPV1 (by 15%), and another cold-sensitive ion channel TRPA1 (by 40%). Only the stimulation of TRPA1 significantly (by 140%) increased antibody formation in the spleen, while TRPM8 had practically no effect on this process, and activation of TRPV1 significantly (by 60%) inhibited antibody formation. Stimulation of the TRPM8 ion channel significantly (by 60%) reduced the level of IgG in the blood, which is believed to control of infectious diseases.The obtained results show that pharmacological activation of the skin TRPA1, TRPM8, TRPV1 ion channels can differently affect the immune system. At the epicenter of changes there were the antigen binding and antibody formation in the spleen, as well as the level of IgG in the blood. Exactly stimulation of the TRPM8 ion channel determines the changes in the immune response when only the skin is cooling, while at deep body heating, the changes in the immune response are mostly determined by the activation of the skin TRPV1 ion channel.

Role of Small Intestine and Gut Microbiome in Plant-Based Oral Tolerance for Hemophilia.

Fusion proteins, which consist of factor VIII or factor IX and the transmucosal carrier cholera toxin subunit B, expressed in chloroplasts and bioencapsulated within plant cells, initiate tolerogenic immune responses in the intestine when administered orally. This approach induces regulatory T cells (Treg), which suppress inhibitory antibody formation directed at hemophilia proteins induced by intravenous replacement therapy in hemophilia A and B mice. Further analyses of Treg CD4+ lymphocyte sub-populations in hemophilia B mice reveal a marked increase in the frequency of CD4+CD25−FoxP3−LAP+ T cells (but not of CD4+CD25+FoxP3+ T cells) in the lamina propria of the small but not large intestine. The adoptive transfer of very small numbers of CD4+CD25−LAP+ Treg isolated from the spleen of tolerized mice was superior in suppression of antibodies directed against FIX when compared to CD4+CD25+ T cells. Thus, tolerance induction by oral delivery of antigens bioencapsulated in plant cells occurs via the unique immune system of the small intestine, and suppression of antibody formation is primarily carried out by induced latency-associated peptide (LAP) expressing Treg that likely migrate to the spleen. Tolerogenic antigen presentation in the small intestine requires partial enzymatic degradation of plant cell wall by commensal bacteria in order to release the antigen. Microbiome analysis of hemophilia B mice showed marked differences between small and large intestine. Remarkably, bacterial species known to produce a broad spectrum of enzymes involved in degradation of plant cell wall components were found in the small intestine, in particular in the duodenum. These were highly distinct from populations of cell wall degrading bacteria found in the large intestine. Therefore, FIX antigen presentation and Treg induction by the immune system of the small intestine relies on activity of a distinct microbiome that can potentially be augmented to further enhance this approach.

Failed B cell survival factor trials support the importance of memory B cells in multiple sclerosis.

Clinical trials are probably the most informative experiments to help an understanding of multiple sclerosis (MS) biology. Recent successes with CD20-depleting antibodies have focused attention towards B cell subsets as important mediators in MS. The trial of tabalumab (NTC00882999), which inhibits B cell activation factor (BAFF), is reported and reviewed and this trial is contrasted with the trial on the inhibition of a proliferation-inducing ligand (APRIL) and BAFF using atacicept (NCT00642902). Both tabalumab and atacicept induce depletion of mature B cells and inhibit antibody formation, but they fail to deplete memory B cells and do not inhibit relapsing MS. Atacicept is reported to augment memory B cell responses and may precipitate relapse, suggesting the importance of APRIL. However, BAFF inhibition can enhance peripheral blood memory B cell responses, which was not associated with augmented relapse. Although other interpretations are possible, these data further support the hypothesis that memory B cells may be of central importance in relapsing MS, as they are the major CD20+ B cell subset expressing APRIL receptors. They also suggest that quantitative and/or qualitative differences in B cell responses or other factors, such as an immune-regulatory effect associated with APRIL, may be important in determining whether MS reactivates following neutralization of peripheral B cell maturation and survival factors.

Immune Modulatory Cell Therapy for Hemophilia B Based on CD20-Targeted Lentiviral Gene Transfer to Primary B Cells

Gene-modified B cells expressing immunoglobulin G (IgG) fusion proteins have been shown to induce tolerance in several autoimmune and other disease models. However, lack of a vector suitable for gene transfer to human B cells has been an obstacle for translation of this approach. To overcome this hurdle, we developed an IgG-human factor IX (hFIX) lentiviral fusion construct that was targeted to specifically transduce cells expressing human CD20 (hCD20). Receptor-specific retargeting by mutating envelope glycoproteins of measles virus (MV)-lentiviral vector (LV) and addition of a single-chain variable fragment specific for hCD20 resulted in gene delivery into primary human and transgenic hCD20 mouse B cells with high specificity. Notably, this protocol neither required nor induced activation of the B cells, as confirmed by minimal activation of inflammatory cytokines. Using this strategy, we were able to demonstrate induction of humoral tolerance, resulting in suppression of antibody formation against hFIX in a mouse model of hemophilia B (HB). In conclusion, transduction of receptor-specific retargeted LV into resting B cells is a promising method to develop B cell therapies for antigen-specific tolerance induction in human disease.

Does everyone with inflammatory bowel disease need to be treated with combination therapy?

This article describes the evolution of combination therapy in inflammatory bowel disease and how to determine when combination may not be necessary. The combination of an immunosuppressive agent with an antibody to tumor necrosis factor has proven to be more effective than monotherapy with either an immunosuppressive agent or an antibody to tumor necrosis factor alone. The emergence of therapeutic drug monitoring has identified the importance of maintaining adequate circulating drug levels and suppressing antibody formation to the biological agent. The concomitant use of an immunosuppressive agent can help effect this. Although this may be optimal therapy in moderately ill patients. I review why there still remains a role for using monotherapy with immunosuppressive agents, as well as consideration of therapy withdrawal. Although combination therapy is the treatment of choice in persons with moderate to severe disease or who have lost response to biological monotherapy, there remains a role for monotherapy with immunosuppressive agents. Though the newer biological therapies are mostly not used in combination, this may be a future approach considering their response rates are also correlated with higher drug levels. Many persons with inflammatory bowel disease can remain well on an immunosuppressive agent alone and some can even maintain a longstanding remission off all therapy.

Albert Coons: harnessing the power of the antibody

Albert Coons: harnessing the power of the antibody

Oral delivery of Acid Alpha Glucosidase epitopes expressed in plant chloroplasts suppresses antibody formation in treatment of Pompe mice.

Deficiency of acid alpha glucosidase (GAA) causes Pompe disease in which the patients systemically accumulate lysosomal glycogen in muscles and nervous systems, often resulting in infant mortality. Although enzyme replacement therapy (ERT) is effective in treating patients with Pompe disease, formation of antibodies against rhGAA complicates treatment. In this report, we investigated induction of tolerance by oral administration of GAA expressed in chloroplasts. Because full-length GAA could not be expressed, N-terminal 410-amino acids of GAA (as determined by T-cell epitope mapping) were fused with the transmucosal carrier CTB. Tobacco transplastomic lines expressing CTB-GAA were generated through site-specific integration of transgenes into the chloroplast genome. Homoplasmic lines were confirmed by Southern blot analysis. Despite low-level expression of CTB-GAA in chloroplasts, yellow or albino phenotype of transplastomic lines was observed due to binding of GAA to a chloroplast protein that has homology to mannose-6 phosphate receptor. Oral administration of the plant-made CTB-GAA fusion protein even at 330-fold lower dose (1.5 μg) significantly suppressed immunoglobulin formation against GAA in Pompe mice injected with 500 μg rhGAA per dose, with several-fold lower titre of GAA-specific IgG1 and IgG2a. Lyophilization increased CTB-GAA concentration by 30-fold (up to 190 μg per g of freeze-dried leaf material), facilitating long-term storage at room temperature and higher dosage in future investigations. This study provides the first evidence that oral delivery of plant cells is effective in reducing antibody responses in ERT for lysosomal storage disorders facilitating further advances in clinical investigations using plant cell culture system or in vitro propagation.

Suppression of Inhibitor Formation in Protein and Gene Therapy for Hemophilia Using Ex Vivo Expanded Treg

Abstract 13Hemophilia A and B result from deficiency in clotting factor VIII (FVIII) or IX (FIX), respectively. In a subset of patients, treatment by factor replacement therapy is limited by formation of inhibitory antibodies to the clotting factors, representing a serious complication that increases risks of morbidity and mortality. Immune responses to the therapeutic coagulation factors are also a concern in newly emerging gene therapies. Regulatory T cells (Treg) offer to be a novel alternative pathway toward immune tolerance. Treg has been identified as a major component of immune tolerance to coagulation factors in pre-clinical studies. Therefore, we hypothesize that ex vivo expanded autologous Treg can suppress inhibitor formation. Our study seeks to test this approach in hemophilic mice. Initially, we optimized in vitro expansion of murine BALB/c-derived Treg. Using flow sorting, GFP+ cells were purified (>98% purity) from spleens of BALB/c knock-in mice containing a GFP reporter linked to FoxP3 expression with an IRES sequence. Sorted cells were stimulated in culture using anti-CD28/anti-CD3 beads in the presence of high-level IL-2 (1000 U/ml). IL-2 was replenished every second day in culture. After ∼1 week, cells were freshly stimulated. At the end of 2 weeks, viability, purity, and FoxP3/GFP expression was confirmed. Greater than 30-fold expansion was repeatedly accomplished. Assumming a dose of 1×106 Treg/mouse, expansion is sufficiently robust to treat >30 mice starting with Treg from 2–3 donor mice. Ex vivo expanded Tregs were adoptively transferred to male hemophilia A mice (BALB/c F8e16 −/−), which were then treated with F.VIII (1 IU human B domain-deleted F.VIII, IV, once per week) for two months. Bethesda assays demonstrated that Treg transplant had effectively suppressed inhibitor formation. Inhibitor titers in control mice were 15–20 BU at 1 month and 30–40 BU at two months. In contrast, Treg treated mice (n=5 per group) formed at most low-titer inhibitors (2–3 BU for both time points). By 2 months, peripheral Treg frequencies had returned to near baseline. To further demonstrate presence of a Treg population capable of suppressing antibody formation against F.VIII, a secondary transfer of sorted CD4+CD25+ splenocytes was performed. Recipient hemophilia A mice were immunized against F.VIII in adjuvant. Compared to mice receiving control Treg, there was significant (P<0.05) suppression of inhibitor formation against F.VIII. In other experiments, Treg therapy was also able to significantly reduce inhibitor titers in hemophilia A mice with pre-existing F.VIII inhibitors. We chose hemophilia A mice (n=6) that developed on average 25 BU from F.VIII replacement therapy. Half of these received Treg transplant, and all mice received 8 more weeks of F.VIII treatment. Inhibitor titers in the control group increased to ∼100 BU. Treg therapy substantially reduced this response (to 15–20 BU, P<0.001). In order to evaluate Treg therapy for hemophilia B, BALB/c F9 −/− × FoxP3-GFP mice were generated. Treg were isolated from these mice as described above, expanded in vitro, and transferred to (BALB/c F9 −/−) hemophilia B mice. Two days later, to test their effectiveness in a gene therapy setting, these mice were treated by intramuscular injection of AAV1 vector expressing human F.IX. By 6 weeks after gene transfer, control mice had formed high-titer antibody against hF.IX (>20 mg IgG/ml plasma, ∼ 10 BU). In contrast, anti-hF.IX formation was undetectable in mice that had received Treg prior to vector administration (n=4/group). While perhaps not as potent as antigen-specific Treg, our data demonstrate the ability of highly purified and ex vivo expanded bulk Treg to control inhibitor formation and thus support their utility for tolerance induction in hemophilia. Their effectiveness may involve emergence of a more specific Treg population after repeated in vivo exposure to antigen. In gene therapy, Treg transplant may be a more desirable alternative to use of immune suppressive drugs. Providing additional immune regulation around the time of vector administration, i.e. when activation signals are provided to the immune system, could be sufficient to prevent immune rejection long-term while inducing antigen-experienced Treg for durable tolerance. Disclosures:Herzog:Genzyme Corp.: Royalties, AAV-FIX technology, Royalties, AAV-FIX technology Patents & Royalties.

Dibenzo[def,p]Chrysene (DBC) Suppresses Antibody Formation in Spleen Cells Following Oral Exposures of Mice

Dibenzo[def,p]chrysene (DBC) is a potent environmental carcinogen in rodents, fish, and human cells examined in culture. There are numerous similarities between the patterns of cytochrome P-450 (P450) activation of DBC and its covalent binding to DNA and proteins with another polycyclic aromatic hydrocarbon (PAH), 7,12-dimethylbenz[a]anthracene (DMBA). Our lab has previously shown that DMBA produces immunosuppression in rodents and human cell systems. Therefore, the purpose of these studies was to examine the immunotoxicity of DBC in a rodent model that was found to be sensitive to the immunosuppressive effects of DMBA. Data showed that DBC had similar potency to DMBA in producing suppression of a T-dependent antibody response (TDAR) and altered spleen cell subsets in a similar manner as DMBA when DMBA was given by gavage for 5 d in corn oil to mice at doses of 1–100 mg/kg total cumulative doses. T-cell-independent antigen (TNP-Ficoll) responses were quantitatively less sensitive to DBC suppression. It was also found that as with DMBA, DBC produced a persistent immunosuppression, which lasted for at least 4 wk following dosing with a novel pill method for self-administration of DBC. In conclusion, DBC appears to possess many of the same characteristics of DMBA in terms of its immunotoxicity.

The influence of TRPM8 ion channel activation on immune response at different temperature conditions

In experiments on rats it was shown that it is possible to modulate the immune response in a whole organism by activating cold-sensitive TRPM8 ion channel by its agonist menthol. The most pronounced changes in the conditions without external temperature stimulation were related to immune parameters for the spleen cells and immunoglobulin level in blood: the activation of TRPM8 ion channel by menthol enhances antigen binding and inhibits antibody formation in spleen, significantly reduces the level of IgG in blood. Activation of TRPM8 ion channel changes the effect of subsequent temperature exposure—cooling or heating. Preliminary application of menthol eliminates the inhibitory effect of deep cooling on immune response. Stimulation of the antigen binding in spleen at deep heating is inversed to suppression in case of heating on the background of TRPM8 activation by menthol. On the contrary, suppression of antibody formation caused by deep heating is eliminated if heating is carried out on the background of TRPM8 stimulation.It can be suggested that a significant influence of TRPM8 agonist menthol on the immune response is mainly related to changes in signal of the skin temperature afferent nerve fibers where TRPM8 ion channel is expressed.

Induction of tolerance to factor VIII by transient co‐administration with rapamycin

Formation of inhibitory antibodies is a frequent and serious complication of factor (F) VIII replacement therapy for the X-linked bleeding disorder hemophilia A. Similarly, hemophilia A mice develop high-titer inhibitors to recombinant human FVIII after a few intravenous injections. Using the murine model, the study sought to develop a short regimen capable of inducing tolerance to FVIII. A 1-month immunomodulatory protocol, consisting of FVIII administration combined with oral delivery of rapamycin, was developed. The protocol effectively prevented formation of inhibitors to FVIII upon subsequent intravenous treatment (weekly for 3.5 months). Control mice formed high-titer inhibitors and had CD4(+) T effector cell responses characterized by expression of IL-2, IL-4 and IL-6. Tolerized mice instead had a CD4(+)CD25(+)FoxP3(+) T cell response to FVIII that suppressed antibody formation upon adoptive transfer, indicating a shift from Th2 to Treg if FVIII antigen was introduced to T cells during inhibition with rapamycin. CD4(+) T cells from tolerized mice also expressed TGF-β1 and CTLA4, but not IL-10. The presence of FVIII antigen during the time of rapamycin administration was required for specific tolerance induction. The study shows that a prophylactic immune tolerance protocol for FVIII can be developed using rapamycin, a drug that is already widely in clinical application. Immune suppression with rapamycin was mild and highly transient, as the mice regained immune competence within a few weeks.

Additional antibody suppression from rituximab added to conventional therapy in severe, refractory anti-GBM nephritis.

Sir, Anti-glomerular basement membrane (GBM) nephritis is an autoimmune disease characterized by IgG antibodyformation against the α3 (IV)NCI non-collagenous region of type IV collagen resulting in rapid progressive glomerulonephritis and, ultimately, renal failure when left untreated [1]. Several novel agents have been used when conventional therapy fails to suppress antibody formation. We describe the case of a young woman with severe anti-GBM disease who had persistently high antibody titres despite conventional therapy, experiencing successful antibody suppression after rituximab was added to her therapeutic regimen. A 20-year-old woman presented at the emergency department with acute, oliguric renal failure due to glomerulonephritis. Creatinine level was 624 mmol/L, urea level was 16 mmol/L, and 24-h protein excretion was 6.2 g. Anti-GBM titre was positive with 270 units, while ANCA-P3/MPO and ANA were negative. The diagnosis of anti-GBM glomerulonephritis was made, and plasmapheresis was initiated while the patient was started on methylprednisolone 1 g i.v. for 3 days followed by 1 mg/kg and cyclophosphamide 2 mg/kg, both orally. At Day 21, after 17 sessions of plasmapheresis with 3 L plasma volumes, the anti-GBM titre remained elevated at 180 units, and proteinuria worsened to 40 g/day. Rituximab was initiated at a dose of 375 mg/m2 with a 2-week interval. Four days after first rituximab treatment, the anti-GBM titre dropped to 130 units, and 11 days later the titre had further decreased to 85 units. Four weeks after the first dose of rituximab and 2 months after the diagnosis was made, the anti-GBM titre had dropped to below reference level, while proteinuria declined to 2 g/24 h and creatinine clearance rose to 32 (Modification of Diet in Renal Disease formula). Phenotyping of peripheral lymphocytes showed B cells to be almost completely absent. The use of rituximab in anti-GBM nephritis has only been described in case studies, with mixed outcome, but its use has been successfully studied as adjuvant or salvage therapy in antibody-mediated autoimmune diseases such as autoimmune thrombocytopenic purpura, autoimmune haemolytic anaemia, systemic lupus erythematosus and rheumatoid arthritis. [2–4] The production of antibody depends on the antigenically driven clonal expansion of B cells into antibody-producing effector plasma cells. Plasma cells are short-lived cells, and survival of the populations depends on differentiation from their progenitor B cells [5]. B-cell ablation with rituximab could prevent further plasma cell and subsequent antibody formation, extinguishing the inflammatory anti-GBM response. In our case, the anti-GBM titre did not decrease below reference level even though 51 L of plasma volume had been exchanged, suggesting a high rate of antibody formation. The rapid decline of antibody titre and proteinuria could very well have been due to a late effect of simultaneous earlier therapy, but the theory behind the use of rituximab does not rule out that the rituximab might have had beneficial effect on antibody formation. Conflict of interest statement. None declared.

Nociceptin/Orphanin FQ Suppresses Adaptive Immune Responses In Vivo and at Picomolar Levels In Vitro

Nociceptin/orphanin FQ (N/OFQ), added in vitro to murine spleen cells in the picomolar range, suppressed antibody formation to sheep red blood cells in a primary and a secondary plaque-forming cell assay. The activity of the peptide was maximal at 10(-12) M, with an asymmetric U-shaped dose-response curve that extended activity to 10(-14) M. Suppression was not blocked by pretreatment with naloxone. Specificity of the suppressive response was shown using affinity-purified rabbit antibodies against two N/OFQ peptides and with a pharmacological antagonist. Antisera against both peptides were active, in a dose-related manner, in neutralizing N/OFQ-mediated immunosuppression, when the peptide was used at concentrations from 10(-12.3) to 10(-11.6) M. In addition, nociceptin given in vivo by osmotic pump for 48 h suppressed the capacity of spleen cells placed ex vivo to make an anti-sheep red blood cell response. These studies show that nociceptin directly inhibits an adaptive immune response, i.e., antibody formation, both in vitro and in vivo.

Effect of anthelmintic Aversect-2 on antibody formation after vaccinating sheep against leptospirosis

It is established that the anthelmintic Aversect-2 suppresses antibody formation after vaccinating sheep against leptospirosis. With simultaneous immunization with polyvalent second variant VGNKI vaccine and dehelminthization, antibodies are elaborated in sheep only to certain serogroups of leptospires.

Perspectives from B cell immunology: fact and fancy

In this article, the formation of antibodies during enzyme replacement therapy (ERT) for lysosomal storage diseases (LSDs) is reviewed in the light of present-day immunological concepts of immunogenicity and tolerance. Except in Gaucher disease, anti-enzyme antibodies frequently form (mainly immunoglobulin G) in patients receiving ERT, though they tend to wane as treatment continues. If the therapeutic enzyme is inhibited by antibodies, no significant modification to treatment is normally warranted, in clear contrast to therapy of hemophilia with clotting factors. The main adverse consequences of ERT, observed in only some patients, are sporadic hypersensitivity reactions, which are likely to be humorally mediated. Some infusion-related reactions are probably due to antibodies. In order to minimize immunogenicity, infused enzymes should be deaggregated and administered at low doses. In addition, inadvertent exposure to co-stimuli that might activate antigen-specific T or B lymphocytes should be avoided. The presence of cross-reacting immunological material, such as in patients with low levels or missense mutations of a gene coding for a lysosomal enzyme, tends to correlate with immune tolerance to the administered enzyme. There is a need for reliable biomarkers for therapeutic efficacy: some directions for further exploration are suggested. In animal models of LSDs, gene therapy delivered via viral vectors can rectify the lysosomal defect, and regulatory T cells that suppress antibody formation can be induced. This is a promising strategy that warrants further investigation in patients with LSDs.

Beneficial Therapeutic Effect of Cellcept® in Systemic Lupus Erythematosis (SLE); Suggested Mechanism of Action (50.26)

Abstract Cellcept® inhibits proliferative responses of T- and B-lymphocytes to both mitogenic and allospecific stimulation and suppresses antibody formation in B-lymphocytes. MPA prevents the glycosylation of lymphocyte and monocyte glycoproteins that are involved in adhesion to endothelial cells. Although Cellcept® is prescribed "off label" to treat SLE and shows great benefits, the exact mechanism of action is not fully understood. MRL/lpr developing glomerular disease mice that exhibit similar pathology to human SLE were used to study the therapeutic effects of Cellcept®. Although Cellcept® at 100mg/kg doses significantly decreased the proteinurea levels in the treated mice, it had marginal effects on the ds-DNA antibody levels. In addition, Cellcept® reduced the levels of B cells in the spleens of treated mice consistent with its immunosuppressant benefit. Histopathological evaluation demonstrated that Cellcept® reduced glomerulonephritis but not vasculitis in kidneys of treated mice. Furthermore, it was shown that Cellcept® lowered the number of monocytes in kidneys; this is the most likely mechanism of action responsible for the less kidney damage and lower proteinurea levels. The reduction in the number of monocytes in kidneys is the first major indication that the beneficial effect of Cellcept® to the kidney function in patients with SLE might be due to the inhibition of mocnocyte infiltration responsible for the destruction of nephritic tissues and not through the action on B cells by lowering the ds-DNA antibody levels.

Emerging Role of Regulatory T Cells in Gene Transfer

Induction and maintenance of immune tolerance to therapeutic transgene products are key requirements for successful gene replacement therapies. Gene transfer may also be used to specifically induce immune tolerance and thereby augment other types of therapies. Similarly, gene therapies for treatment of autoimmune diseases are being developed in order to restore tolerance to self-antigens. Regulatory T cells have emerged as key players in many aspects of immune tolerance, and a rapidly increasing body of work documents induction and/or activation of regulatory T cells by gene transfer. Regulatory T cells may suppress antibody formation and cytotoxic T cell responses and may be critical for immune tolerance to therapeutic proteins. In this regard, CD4(+)CD25(+) regulatory T cells have been identified as important components of tolerance in several gene transfer protocols, including hepatic in vivo gene transfer. Augmentation of regulatory T cell responses should be a promising new tool to achieve tolerance and avoid immune-mediated rejection of gene therapy. During the past decade, it has become obvious that immune regulation is an important and integral component of tolerance to self-antigens and of many forms of induced tolerance. Gene therapy can only be successful if the immune system does not reject the therapeutic transgene product. Recent studies provide a rapidly growing body of evidence that regulatory T cells (T(reg)) are involved and often play a crucial role in tolerance to proteins expressed by means of gene transfer. This review seeks to provide an overview of these data and their implications for gene therapy.