The Influence of Glycans-Specific Bioconjugation on the FcγRI Binding and In vivo Performance of 89Zr-DFO-Pertuzumab.

Rationale: The overwhelming majority of radioimmunoconjugates are produced via random conjugation methods predicated on attaching bifunctional chelators to the lysines of antibodies. However, this approach inevitably produces poorly defined and heterogeneous immunoconjugates because antibodies have several lysines distributed throughout their structure. To circumvent this issue, we have previously developed a chemoenzymatic bioconjugation strategy that site-specifically appends cargoes to the biantennary heavy chain glycans attached to CH2 domains of the immunoglobulin's Fc region. In the study at hand, we explore the effects of this approach to site-specific bioconjugation on the Fc receptor binding and in vivo behavior of radioimmunoconjugates. Methods: We synthesized three desferrioxamine (DFO)-labeled immunoconjugates based on the HER2-targeting antibody pertuzumab: one using random bioconjugation methods (DFO-nsspertuzumab) and two using variants of our chemoenzymatic protocol (DFO-sspertuzumab-EndoS and DFO-sspertuzumab-ßGal). Subsequently, we characterized these constructs and evaluated their ability to bind HER2, human FcγRI (huFcγRI), and mouse FcγRI (muFcγRI). After radiolabeling the immunoconjugates with zirconium-89, we conducted PET imaging and biodistribution studies in two different mouse models of HER2-expressing breast cancer. Results: MALDI-ToF and SDS-PAGE analysis confirmed the site-specific nature of the bioconjugation, and flow cytometry and surface plasmon resonance (SPR) revealed that all three immunoconjugates bind HER2 as effectively as native pertuzumab. Critically, however, SPR experiments also illuminated that DFO-sspertuzumab-EndoS possesses an attenuated binding affinity for huFcγRI (17.4 ± 0.3 nM) compared to native pertuzumab (4.7 ± 0.2 nM), DFO-nsspertuzumab (4.1 ± 0.1 nM), and DFO-sspertuzumab-ßGal (4.7 ± 0.2 nM). ImmunoPET and biodistribution experiments in athymic nude mice bearing HER2-expressing BT474 human breast cancer xenografts yielded no significant differences in the in vivo behavior of the radioimmunoconjugates. Yet experiments in tumor-bearing humanized NSG mice revealed that 89Zr-DFO-sspertuzumab-EndoS produces higher activity concentrations in the tumor (111.8 ± 39.9 %ID/g) and lower activity concentrations in the liver and spleen (4.7 ± 0.8 %ID/g and 13.1 ± 4.0 %ID/g, respectively) than its non-site-specifically labeled cousin, a phenomenon we believe stems from the altered binding of the former to huFcγRI. Conclusion: These data underscore that this approach to site-specific bioconjugation not only produces more homogeneous and well-defined radioimmunoconjugates than traditional methods but may also improve their in vivo performance in mouse models by reducing binding to FcγRI.

Efficacy of Bortezomib as an Adjunctive Therapy for Refractory Chronic Active Antibody-Mediated Rejection in Kidney Transplant Patients: A Single-Center Experience.

BACKGROUND: Chronic active antibody-mediated rejection (CAMR) has unsatisfactory prognosis in spite of intensive standard antihumoral treatment. Efficacy of additional bortezomib in CAMR remains uncertain. METHODS: A retrospective chart review was conducted among kidney transplant patients with biopsy-proven CAMR. Our standard CAMR protocol included plasma exchange, intravenous immunoglobulin, and rituximab. Repeated treatment was provided for refractory cases. Patients receiving at least 1 course of bortezomib were enrolled as the bortezomib group. Allograft outcome was compared among patients receiving repeated standard protocol alone and the bortezomib group. RESULTS: Thirteen and 15 patients were assigned to the bortezomib and control groups, respectively. Repeated bortezomib protocol was given for 1, 2, 3, and 4 courses in 6, 4, 1, and 2 patients, respectively. With a median follow-up time after treatment of 41.8 (18.3-47.4) months, the bortezomib group had a lower rate of glomerular filtration rate declination (-4.20 ± 4.89 mL/min/y vs -12.33 ± 10.44 mL/min/y; P = .014), a higher rate of disappearance of donor specific antibodies (69.2% vs 25%; P = .03), a lower rate of allograft loss (15.4% vs 66.7%; P = .006), and better allograft survival (P = .006). CONCLUSION: In CAMR, additional bortezomib treatment was more effective in eliminating donor specific antibodies and improving allograft survival than standard protocol treatment.

[Fam-] trastuzumab deruxtecan (DS-8201a)-induced antitumor immunity is facilitated by the anti-CTLA-4 antibody in a mouse model.

[Fam-] trastuzumab deruxtecan (DS-8201a) is a HER2 (ERBB2)-targeting antibody-drug conjugate, composed of a HER2-targeting antibody and a topoisomerase I inhibitor, exatecan derivative, that has antitumor effects in preclinical xenograft models and clinical trials. Recently, [fam-] trastuzumab deruxtecan was reported to enhance antitumor immunity and was beneficial in combination with an anti-PD-1 antibody in a mouse model. In this study, the antitumor effect of [fam-] trastuzumab deruxtecan in combination with an anti-CTLA-4 antibody was evaluated. [Fam-] trastuzumab deruxtecan monotherapy had antitumor activity in an immunocompetent mouse model with EMT6 human HER2-expressing mouse breast cancer cells (EMT6-hHER2). [Fam-] trastuzumab deruxtecan in combination with the anti-CTLA-4 antibody induced more potent antitumor activity than that by monotherapy with either agent. The combination therapy increased tumor-infiltrating CD4+ and CD8+ T cells in vivo. Mechanistically, cured mice with treatment of [fam-] trastuzumab deruxtecan and an anti-CTLA-4 antibody completely rejected EMT6-mock cells similar to EMT6-hHER2 cells, and splenocytes from the cured mice responded to both EMT6-hHER2 and EMT6-mock cells as measured by interferon-gamma release. Taken together, these results indicate that antitumor immunity is induced by [fam-] trastuzumab deruxtecan and is facilitated in combination with anti-CTLA-4 antibody.

Hypofractionated EGFR tyrosine kinase inhibitor limits tumor relapse through triggering innate and adaptive immunity.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are a first-line therapy for rapidly killing tumors such as those associated with non-small cell lung cancer by blocking oncogenic receptor signaling, but tumor relapse often occurs. Here, we have observed that hypofractionated EGFR TKI treatment (HypoTKI) is more potent than standard hyperfractionated EGFR TKI treatment (HyperTKI), and its antitumor effect associated with preventing tumor relapse depends on T cells. HypoTKI triggers greater innate sensing for type I IFN and CXCL10 production through the Myd88 signaling pathway to enhance tumor-specific T cell infiltration and reactivation. We also demonstrate that timely programmed cell death ligand-1 (PD-L1) blockade can synergize with HypoTKI to control advanced large tumors and effectively limit tumor relapse without severe side effects. Our study provides evidence for exploring the potential of a proper combination of EGFR TKIs and immunotherapy as a first-line treatment for treating EGFR-driven tumors.

Outcome of Desensitization Therapy in Immunologically High-Risk Kidney Transplantation: Single-Center Experience.

AIM: Sensitization to HLA antigens creates an immunologic barrier, linked to an increased risk of antibody-mediated rejection and poorer graft survival, that remains a persistent and often impenetrable deterrent to transplantation. Desensitization can improve transplantation rates in broadly sensitized kidney transplant recipients. We aimed to compare the clinical outcomes of immunologic high-risk kidney recipients who had desensitization treatment with the outcomes of those who did not. MATERIALS AND METHODS: We retrospectively evaluated patients who underwent desensitization protocol due to immunologic risk between 2010 and 2018. Living-donor transplantation patients with panel reactive antibody positivity, retransplantation, donor specific antibody, and/or single antigen bead positivity were included in the study. We excluded deceased-donor transplantation recipients. Demographic data (age, sex, etiology of end-stage renal disease, blood transfusions, pregnancy, etc), immunologic status (HLA-mismatch [HLA-MM], panel reactive antibody, donor specific antibody, etc), induction and maintenance of immunosuppressive medications, and complications (all-cause hospitalizations, episodes of acute rejections, etc) were noted. We compared data and clinical outcomes of patients who had desensitization (Group 1) with data and clinical outcomes of patients who had not had desensitization (Group 2). FINDINGS: There were 124 living-kidney donors (49 female, mean age 43.7 ± 12.2 years, mean body mass index [BMI] 25.8 ± 5.8 kg/m2, mean follow-up time 20.9 ± 14.6 months). Thirty-four of these patients (25 female, mean age 43.7 ± 12.5 years, mean follow-up time 26.1 ± 17.7 months, mean BMI 27 ± 6.5 kg/m2) had desensitization treatment (rituximab+plasmapheresis for 19 patients, rituximab for 11 patients, rituximab+plasmapheresis+intravenous immunoglobulin for 4 patients). Ninety patients (24 female, mean age 43.7 ± 12.2 years, mean follow-up time 18.9 ± 12.9 months, mean BMI 25.3 ± 5.4 kg/m2) had not had desensitization. There was no statistical difference between groups for age, sex, hepatitis serology, history of blood transfusion, history of pregnancy, or history of dialysis (P < .05 for all parameters). While scores for HLA-MM and HLA-relative intensity scale (RIS) were 2.7 ± 1.6 and 7.86 ± 6.2, respectively, in Group 1, in Group 2 the same scores were 2.1 ± 1.1 and 3.6 ± 2.5, respectively (P: .053 and .03). Delayed graft function, acute rejection episodes, and hospitalizations were similar between groups (P: .47, .29, and .34, respectively). Follow-up time and length of hospitalization were longer in Group 1 (P: .013 and .001, respectively). Total doses of ATG were higher in Group 1 patients (P: .007). CONCLUSION: Despite the higher HLA-MM and RIS scores, clinical outcomes in desensitized patients were found to be similar to those in nondesensitized patients for acute rejection episodes and hospitalizations. Desensitization with rituximab in patients with high HLA-RIS scores can prevent acute rejection and hospitalization.

Evaluación de la actividad inmunomoduladora del polvo seco de Punica granatum Linn en un modelo experimental de inmunosupresión / Evaluation of the immunomodulatory activity of dry powder of Punica granatum Linn in an experimental model of immunosuppression

Introducción: La inmunosupresión es uno de los principales obstáculos en el tratamiento de cáncer, por esta razón, diversos inmunomoduladores naturales y sintéticos son estudiados, con el fin de atenuar los efectos de la terapia convencional. Objetivo: Evaluar la actividad inmunomoduladora del polvo seco de Punica granatum Linn (granada). Métodos: Ratas machos Wistar fueron divididas en cuatro grupos: grupo I (control), II (inmunizado con glóbulos rojos de carnero), III (inmunosuprimido con ciclofosfamida e inmunizado con glóbulos rojos de carnero) y IV (tratado con polvo de granada, inmunosuprimido con ciclofosfamida e inmunizado con glóbulos rojos de carnero). Las variables estudiadas fueron el peso corporal y detección de anticuerpos hemoaglutinantes. Resultados: Se observó ligera tendencia al aumento del peso corporal en los grupos I, II y III, con respuesta diferente en el grupo IV, donde hubo una leve disminución. El título de anticuerpos del grupo III disminuyó con respecto al II y IV, tanto en la respuesta primaria como secundaria. En el grupo IV el título de anticuerpos resultó ser estadísticamente significativo con relación al del grupo III (p= 0,000) para ambas respuestas. Conclusiones: La Púnica granatum mostró efecto inmunomodulador, al incrementar el nivel de anticuerpos hemaglutinantes(AU)

Introduction: Immunosuppression is one of the main obstacles in the treatment of cancer, for this reason, several natural and synthetic immunomodulators are studied, in order to attenuate the effects of conventional therapy. Objective: To evaluate the immunomodulator activity of Punica granatum Linn (pomegranade). Methods: Wistar male rats were divided into four groups: group I (control), II (immunized with red blood cells of sheep), III (immunosuppressed with cyclophosphamide and immunized with red blood cells of sheep) and IV (treated with pomegranate powder, immunosuppressed with cyclophosphamide and immunized with red blood cells of sheep). The variables studied were body weight and detection of haemagglutinating antibodies. Results: A slight tendency to increase body weight was observed in groups I, II and III, with a different response in group IV, where there was a slight decrease. The antibody titre of group III decreased with respect to II and IV, both in the primary and secondary response. In group IV the antibody titer was found to be statistically significant in relation to group III (p = 0.000) for both responses. Conclusions: Punic granatum showed immunomodulatory effect, increasing the level of haemagglutinating antibodies(AU)

HLA-DRB3*01:01 exhibits a dose-dependent impact on HPA-1a antibody levels in HPA-1a-immunized women.

HLA-DRB3*01:01 is a predisposing factor for human platelet antigen 1a (HPA-1a) immunization, which is responsible for most cases of fetal and neonatal alloimmune thrombocytopenia. The aim of this study was to investigate if the HLA-DRB3*01:01 allele imposes a dose-dependent effect on anti-HPA-1a levels and neonatal platelet counts. One hundred and thirty HPA-1a-immunized women were divided into 3 groups: HLA-DRB3*01:01 negative, HLA-DRB3*01:01 hemizygous or heterozygous, and HLA-DRB3*01:01 homozygous. The dose of the HLA-DRB3*01:01 allele was determined by sequencing exon 2 of the HLA-DRB3 gene followed by HLA-DRB3 and HLA-DRB1 typing of selected samples. Anti-HPA-1a levels at time of delivery and neonatal platelet counts were compared among groups. There was a significant dose-dependent effect of the HLA-DRB3*01:01 allele on anti-HPA-1a levels (global P value [P global] = .0032). Median (range) anti-HPA-1a levels were 1.5 IU/mL (0.0-19.0 IU/mL), 21.1 IU/mL (0.0-1967 IU/mL), and 43.7 IU/mL (1.0-980 IU/mL) in women with 0, 1, and 2 copies of the HLA-DRB3*01:01 allele, respectively. There was also a significant, but opposite, dose-dependent effect of the mother's HLA-DRB3*01:01 allele on the platelet count of the newborn (P global = .0155). Median (range) neonatal platelet counts were 241 × 109/L (59 × 109/L to 393 × 109/L), 107 × 109/L (4 × 109/L to 387 × 109/L) and 32 × 109/L (4 × 109/L to 352 × 109/L) for newborns of mothers with 0, 1, and 2 copies of the HLA-DRB3*01:01 allele, respectively. Thus, the HLA-DRB3*01:01 allele exhibits a dose-dependent impact on maternal anti-HPA-1a levels in HPA-1a-immunized women.

Longitudinal monitoring of anti-saliva antibodies as markers of repellent efficacy against Phlebotomus perniciosus and Phlebotomus papatasi in dogs.

A 2-year longitudinal study of enzyme-linked immunosorbent assay (ELISA) antibodies against Phlebotomus perniciosus and Phlebotomus papatasi (Diptera: Psychodidae) sandfly saliva was performed in 32 Beagle dogs treated preventively with an imidacloprid-permethrin topical insecticide in an endemic area in Spain. Dogs were grouped into three sandfly exposure groups according to the time of inclusion in the study. Assays analysed immunoglobulin G (IgG) against salivary gland homogenates (SGH) of both species and recombinant P. papatasi rSP32 and P. perniciosus rSP03B proteins in serum. The dogs were participating in a Leishmania infantum (Kinetoplastida: Trypanosomatidae) vaccine trial and were experimentally infected with the parasite in the second year. No dog acquired natural L. infantum infections during the first year, but most developed anti-saliva antibodies, and median log-transformed optical densities (LODs) were seasonal, mimicking those of local sandflies. This indicates that the repellent efficacy of the insecticide used is below 100%. Multi-level modelling of LODs revealed variability among dogs, autocorrelation and differences according to the salivary antigen and the dog's age. However, dog seroprevalence, estimated using pre-exposure LODs as cut-offs, was relatively low. This, and the fact that dogs did not become naturally infected with L. infantum, would support the efficacy and usefulness of this imidacloprid-permethrin topical insecticide in canine leishmaniasis control.

Quantitative Assessment of the Effects of Reducing Agents on Biological Macromolecules and on the Possible Repair of Oxidative Damage.

OBJECTIVE: To quantitatively assess the influence of reducing agents on biological macromolecules and on the possible repair of oxidative damage. METHODS: Samples (antibody, enzyme, DNA, and diluted serum) were treated with reducing agents (ammonium ferrous sulfate, ascorbic acid, potassium iodide, and sodium hyposulfite) in the experimental group and with NaCl in the control group. Enzyme-linked immunosorbent assay and quantitative PCR were used to determine the activity of antibody, enzyme, and DNA. Native gel electrophoresis (Native-PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to determine protein structure. Reducing agents that had no inhibitory effect on biological macromolecules were selected. Antibodies were treated with oxidants to caused oxidative damage and then treated with reducing agents, and the possible repair of oxidative damage was assessed. RESULTS: Certain concentrations of ammonium ferrous sulfate resulted in significant inhibition of antibody, enzyme, DNA, and diluted serum. Certain concentrations of ascorbic acid resulted in significant inhibition of antibody. Sodium hyposulfite and potassium iodide had no effect on antibody, enzyme, DNA, and diluted serum. The OD values in group A (in which HBsAb was treated by oxidation and then a reductant) were significantly higher than those in group B (HBsAb treated by oxidation). CONCLUSION: Ammonium ferrous sulfate, ascorbic acid, sodium hyposulfite, and potassium iodide had different effects on antibody, enzyme, DNA, and diluted serum. The reduction in antibody activity due to an oxidant was partially repaired by a reductant.

Antibodies targeting surface membrane antigens in patients with chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplant reflects a complex immune response resulting in chronic damage to multiple tissues. Previous studies indicated that donor B cells and the antibodies they produce play an important role in the development of cGVHD. To understand the pathogenic role of antibodies in cGVHD, we focused our studies on posttransplant production of immunoglobulin G antibodies targeting cell surface antigens expressed in multiple cGVHD affected tissues, due to their potential functional impact on living cells in vivo. Using plate-bound cell membrane proteins as targets, we detected a significantly higher level of antibodies reactive with these membrane antigens in patients who developed cGVHD, compared with those who did not and healthy donors. Plasma-reactive antibody levels increased significantly prior to the clinical diagnosis of cGVHD and were reduced following cGVHD therapies including prednisone, interleukin-2, or extracorporeal photophoresis. Using cell-based immunoprecipitation with plasma from cGVHD patients and mass spectrometry, we identified 43 membrane proteins targeted by these antibodies. The presence of antibodies in cGVHD patients' plasma that specifically target 6 of these proteins was validated. Antibodies reactive with these 6 antigens were more frequently detected in patients with cGVHD compared with patients without cGVHD and healthy donors. These results indicate that antibodies that target membrane antigens of living cells frequently develop in cGVHD patients and further support a role for B cells and antibodies in the development of cGVHD.

Encapsulating an Immunosuppressant Enhances Tolerance Induction by Siglec-Engaging Tolerogenic Liposomes.

Unwanted antibody responses significantly impact human health, and current options for treating deleterious antibody responses largely rely on broad immunosuppressants that can compromise overall immunity. A desirable alternative is to induce antigen-specific immune tolerance. We have shown that co-presentation of antigen and ligands of B cell sialic acid-binding immunoglobulin-like lectins (Siglecs) on a liposomal nanoparticle induces antigen-specific tolerance. Although Siglec-engaging tolerance-inducing antigenic liposomes (STALs) induce robust B cell tolerance in naïve mice, the full potential of STALs requires long-term tolerance induction and suppression of an ongoing immune response. We hypothesized that STALs encapsulated with rapamycin (RAPA), an immunomodulator, could improve the efficacy of STALs and potentially enable their use in the context of immunological memory. Here, we showed that formulation of STALs with RAPA produced enhanced tolerance induction in naïve mice compared to STALs without RAPA but had minimal impact on inducing tolerance in previously sensitized mice. These findings indicate that the addition of immunomodulators to STALs could be beneficial in tolerance induction and support future development of STALs for the treatment of allergy and autoimmune diseases.

Cyclodimerization of immunosuppressive fragment of HLA-DR molecule. Design, synthesis and ESI-MS/MS analysis.

The nonapeptide fragment of the HLA-DR molecule, located in the exposed loop of the alpha-chain (164-172), having the VPRSGEVYT sequence, suppresses the immune response. Based on the three-dimensional structure of the HLA-DR superdimer, we designed a new cyclodimeric analog in which the two parallel peptide chains of VPRSGEVYT sequence are linked through their C-termini by spacer of (Gly5 )2 -Lys-NH2 and the N-termini are also linked by poly(ethylene glycol). The (VPRSGEVYTG5 )2 K-resin analog was synthesized using solid-phase peptide synthesis protocols. The cyclization was achieved by cross-linking the N-terminal positions of the dimeric peptide, attached to a MBHA resin, with alpha, omega-bis (acetic acid) poly(ethylene glycol), activated by esterification with pentafluorophenol. Our results demonstrate that the cyclodimerization of VPRSGEVYT results in enhanced immunosuppressive activity of the peptide. Mass spectrometry fragmentation analysis of the obtained cyclodimeric peptide is also presented. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Ephrin-B2 prevents N-methyl-D-aspartate receptor antibody effects on memory and neuroplasticity.

OBJECTIVE: To demonstrate that ephrin-B2 (the ligand of EphB2 receptor) antagonizes the pathogenic effects of patients' N-methyl-D-aspartate receptor (NMDAR) antibodies on memory and synaptic plasticity. METHODS: One hundred twenty-two C57BL/6J mice infused with cerebrospinal fluid (CSF) from patients with anti-NMDAR encephalitis or controls, with or without ephrin-B2, were investigated. CSF was infused through ventricular catheters connected to subcutaneous osmotic pumps over 14 days. Memory, behavioral tasks, locomotor activity, presence of human antibodies specifically bound to hippocampal NMDAR, and antibody effects on the density of cell-surface and synaptic NMDAR and EphB2 were examined at different time points using reported techniques. Short- and long-term synaptic plasticity were determined in acute brain sections; the Schaffer collateral pathway was stimulated and the field excitatory postsynaptic potentials were recorded in the CA1 region of the hippocampus. RESULTS: Mice infused with patients' CSF, but not control CSF, developed progressive memory deficit and depressive-like behavior along with deposits of NMDAR antibodies in the hippocampus. These findings were associated with a decrease of the density of cell-surface and synaptic NMDAR and EphB2, and marked impairment of long-term synaptic plasticity without altering short-term plasticity. Administration of ephrin-B2 prevented the pathogenic effects of the antibodies in all the investigated paradigms assessing memory, depressive-like behavior, density of cell-surface and synaptic NMDAR and EphB2, and long-term synaptic plasticity. INTERPRETATION: Administration of ephrin-B2 prevents the pathogenic effects of anti-NMDAR encephalitis antibodies on memory and behavior, levels of cell-surface NMDAR, and synaptic plasticity. These findings reveal a strategy beyond immunotherapy to antagonize patients' antibody effects. Ann Neurol 2016;80:388-400.

Synthesis and immunopotentiating activity of novel isoxazoline functionalized coumarins.

A novel series (13) of isoxazoline functionalized coumarins was synthesized through 1,3-dipolar cyclization of nitrile oxides with Allylated coumarins. Synthesis of effective and target selective immunostimulators through conjugation of diversely substituted isoxazolines and 7-hydroxycoumarins is the focus of the present article. The proposed synthetic scheme was observed to be highly regiospecific yielding attempted conjugates in good yield (>90%). Kinetic resolution of the racemates was carried out by employing lipase B from Candida antarctica (CALB). The synthesized compounds were screened in vitro and in vivo for their biological activities viz. toxicity and impact on splenocyte proliferation (T- and B-cell proliferation), antibody production (HA titre), delayed-type hypersensitivity reaction (DTH), T-cell subtypes (CD4 and CD8), cytokine production (IL-2, IFN-γ, and IL-4) and NO (macrophage) production. Our results establish that isoxazoline functionalized coumarins exhibit excellent immune potentiating activity especially compounds 2, 4 and 8 whose activity is more than that of Levimasole as standard. The structure activity relations are explained in light of the structural/functional aspects of tested compounds. To the best of our knowledge the presented work is first of its kind and is presaged to prove very useful for the design and synthesis of bis-heterocycle based novel, therapeutically selective and effective immunopotentiators.

[Treatment with rituximab as an opportunity for the prevention of infectious complications]. / Lécba rituximabem jako výzva k prevenci infekcních komplikací.

UNLABELLED: Rituximab, a monoclonal antibody against the surface antigen of B-lymphocytes CD20 is beeing used in the treatment of numerous hematological, hematooncological and autoimmune disorders. After administration of ritu-ximab, quick and almost complete depletion of B-lymphocytes with the exception of pre-B-lymphocytes and plasma cells occur. Neutropenia and low serum antibody levels in classes IgA, IgM and IgG may also develop. These changes usually persist for 6-12 months, rarely for several years. In the consequence, patients with the rituximab treatment are more prone to infections - usually of bacterial and viral origin. Concomitantly, rituximab treatment influences negatively postvaccination antibody production and therefore adequate preventive measures are necessary before the beginning of the treatment. The authors offer complex overview of actual literature, emphasize adequate education of patients as well as of healthcare providing staff and discuss the vaccination recommendation against preventable communicable diseases like influenza, pneumococcal diseases, tetanus, diphtheria and pertussis. KEY WORDS: autoimmune disease - immunosupression - infectious complications - prevention - rituximab - vaccination.

Anti-CD28 Antibody and Belatacept Exert Differential Effects on Mechanisms of Renal Allograft Rejection.

Belatacept is a biologic that targets CD80/86 and prevents its interaction with CD28 and its alternative ligand, cytotoxic T lymphocyte antigen 4 (CTLA-4). Clinical experience in kidney transplantation has revealed a high incidence of rejection with belatacept, especially with intensive regimens, suggesting that blocking CTLA-4 is deleterious. We performed a head to head assessment of FR104 (n=5), a selective pegylated Fab' antibody fragment antagonist of CD28 that does not block the CTLA-4 pathway, and belatacept (n=5) in kidney allotransplantation in baboons. The biologics were supplemented with an initial 1-month treatment with low-dose tacrolimus. In cases of acute rejection, animals also received steroids. In the belatacept group, four of five recipients developed severe, steroid-resistant acute cellular rejection, whereas FR104-treated animals did not. Assessment of regulatory T cell-specific demethylated region methylation status in 1-month biopsy samples revealed a nonsignificant trend for higher regulatory T cell frequencies in FR104-treated animals. Transcriptional analysis did not reveal significant differences in Th17 cytokines but did reveal higher levels of IL-21, the main cytokine secreted by CD4 T follicular helper (Tfh) cells, in belatacept-treated animals. In vitro, FR104 controlled the proliferative response of human preexisting Tfh cells more efficiently than belatacept. In mice, selective CD28 blockade also controlled Tfh memory cell responses to KLH stimulation more efficiently than CD80/86 blockade. Our data reveal that selective CD28 blockade and belatacept exert different effects on mechanisms of renal allograft rejection, particularly at the level of Tfh cell stimulation.

In Contrast to Anti-C5 Therapy, Cobra Venom Factor Does Not Prevent Rejection of Xenogeneic Cartilage in Mice.

BACKGROUND: The use of xenogeneic chondrocytes may benefit the development of clinical tissue-engineering applications for cartilage repair. However, cartilage xenografts are slowly rejected by humoral and cellular mechanisms to which galactose α1,3-galactose (Gal) antigen and complement contribute. Accordingly, transgenic expression of human α1,2-fucosyltransferase (HT) in porcine cartilage helps to protect from the Gal-mediated immune response. Here, we aimed to assess the effect of the broadly used complement inhibitor cobra venom factor (CVF) in comparison with anti-C5 therapy in α1,3-galactosyltransferase knockout (Gal KO) mice transplanted with porcine cartilage. METHODS: Gal KO mice grafted with control or HT-transgenic cartilage were left untreated or treated systemically with either anti-C5 antibody or CVF for 5 weeks. The degree of rejection was evaluated by use of histopathological analysis, and serum anti-Gal antibodies were measured in all cohorts. RESULTS: The rejection process of control cartilage was well advanced by 5 weeks after transplantation in untreated Gal KO mice, whereas enhanced graft survival characterized by reduced cellular immune infiltrate was found in mice grafted with HT cartilage and/or treated with anti-C5. In contrast, CVF administration led to inconsistent results, with some grafts showing no improvement or even increased amounts of granulocytes. Regarding antibody titers, the anti-Gal immunoglobulin (Ig)M increased in the control transplant cohort and remained unchanged in the HT-graft recipients at 5 weeks after transplantation. Notably, a strong anti-Gal IgM response was readily detected in CVF-treated mice of both transplanted cohorts. CONCLUSIONS: CVF does not present advantages over anti-C5 therapy for preventing rejection of xenogeneic porcine cartilage.

Transcutaneous immunization with phosphorylcholine induces antigen-specific mucosal and systemic immune responses in BALB/c mice.

OBJECTIVE: Transcutaneous immunization (TCI) is a novel route of vaccination through application of a topical vaccine antigen on the skin. Phosphorylcholine (PC) is a structural component of a variety of pathogens and anti-PC immune responses protect mice against invasive bacterial diseases. The purpose of the study was to examine the effect of TCI using PC in BALB/c mice. METHODS: TCI was performed in BALB/c mice using PC-keyhole limpet hemocyanin (KLH) plus cholera toxin (CT). Immunogenicity was evaluated by measuring PC-specific IgG and specific IgG1, IgG2a, IgM, IgA, and secretory IgA antibodies by ELISA. The concentrations of IL-4, IL-5, IL-10, IL-12, IL-13 and IFN-γ were also measured using ELISA for mouse. RESULTS: Six months after immunization, IgG after TCI using PC plus CT was significantly higher than in controls, but this was not found for IgA. In saliva, secretory IgA antibodies decreased with a peak level at 2-3 months. IgG1 was significantly higher than IgG2 after TCI. Production of IL-4 from CD4(+) cells was significantly higher after TCI than in controls, whereas production of IFN-γ, IL-5, IL-12 and IL-13 was not detected in either group. CONCLUSION: These results suggest that TCI using PC plus CT with BALB/c mice is a simple approach for induction of systemic and mucosal immune responses that are shifted in the Th-2 direction.