Dwindled serum IgG levels of Rubella, Diphtheria toxin, Hepatitis B virus and Tetanus Toxoid after chemotherapy; a report from Iranian children with malignancy

Backgrounds: Epigenetic regulation such as DNA methylation plays a major role in chromatin organization Background: Chemotherapy suppresses immunoglobulin production as a result of cell toxicity. Decreased immunoglobulin levels can result in the onset of opportunistic infections. The aim of the current study is to compare the immunoglobulin G (IgG) levels of the selected vaccine-preventable disease (VPD) before and six months after chemotherapy in a group of Iranian children with malignancies. Materials and Methods: In this interventional study, serum levels of Rubella, Diphtheria toxin, Hepatitis B virus (HBV), Tetanus Toxoid, Mumps, and Measles IgG were measured in 30 children with malignancy and previously vaccinated for these diseases. Six months after chemotherapy, serum IgG levels were reassessed and compared with their corresponding pre-chemotherapy levels. Results: In this study, 17 (56.7%) male and 13 (43.3%) female were included. The mean age was 7.69±3.09 years. After chemotherapy, Rubella IgG levels dropped from 73.88±85.11 to 56.59±72.84 IU/mL (P<0.05; r= 0.956; 33.4% become serum negative (SN)). Diphtheria toxin IgG was diminished from 0.683±0.454 to 0.174±0.248 IU/mL (P<0.05; r=0.601; 26.7% SN). Anti-HBV IgG showed a reduction from 46.26±101.56 to 25.56±80.49 IU/mL (p<0.05; r= 0.524; 60% SN) and Anti-Tetanus Toxoid IgG fell down from 1.031±0.582 to 0.321±0.408 IU/mL (p<0.05; r= 0.365; 33.4% SN). Anti-Measles and Anti-Mumps IgGs showed no significant change (p>0.05). Conclusion: Pediatric chemotherapy was associated with dropped serum IgG levels of most VPDs. A good correlation was also observed between serum levels of IgG before and six months after chemotherapy. Revaccination of children with malignancies may be necessary upon declined serum IgG titers.

Diphtheria toxin induced but not CSF1R inhibitor mediated microglia ablation model leads to the loss of CSF/ventricular spaces in vivo that is independent of cytokine upregulation

Background Two recently developed novel rodent models have been reported to ablate microglia, either by genetically targeting microglia (via Cx3cr1-creER: iDTR + Dtx) or through pharmacologically targeting the CSF1R receptor with its inhibitor (PLX5622). Both models have been widely used in recent years to define essential functions of microglia and have led to high impact studies that have moved the field forward. Methods Using either Cx3cr1-iDTR mice in combination with Dtx or via the PLX5622 diet to pharmacologically ablate microglia, we compared the two models via MRI and histology to study the general anatomy of the brain and the CSF/ventricular systems. Additionally, we analyzed the cytokine profile in both microglia ablation models. Results We discovered that the genetic ablation (Cx3cr1-iDTR + Dtx), but not the pharmacological microglia ablation (PLX5622), displays a surprisingly rapid pathological condition in the brain represented by loss of CSF/ventricles without brain parenchymal swelling. This phenotype was observed both in MRI and histological analysis. To our surprise, we discovered that the iDTR allele alone leads to the loss of CSF/ventricles phenotype following diphtheria toxin (Dtx) treatment independent of cre expression. To examine the underlying mechanism for the loss of CSF in the Cx3cr1-iDTR ablation and iDTR models, we additionally investigated the cytokine profile in the Cx3cr1-iDTR + Dtx, iDTR + Dtx and the PLX models. We found increases of multiple cytokines in the Cx3cr1-iDTR + Dtx but not in the pharmacological ablation model nor the iDTR + Dtx mouse brains at the time of CSF loss (3 days after the first Dtx injection). This result suggests that the upregulation of cytokines is not the cause of the loss of CSF, which is supported by our data indicating that brain parenchyma swelling, or edema are not observed in the Cx3cr1-iDTR + Dtx microglia ablation model. Additionally, pharmacological inhibition of the KC/CXCR2 pathway (the most upregulated cytokine in the Cx3cr1-iDTR + Dtx model) did not resolve the CSF/ventricular loss phenotype in the genetic microglia ablation model. Instead, both the Cx3cr1-iDTR + Dtx ablation and iDTR + Dtx models showed increased activated IBA1 + cells in the choroid plexus (CP), suggesting that CP-related pathology might be the contributing factor for the observed CSF/ventricular shrinkage phenotype. Conclusions Our data, for the first time, reveal a robust and global CSF/ventricular space shrinkage pathology in the Cx3cr1-iDTR genetic ablation model caused by iDTR allele, but not in the PLX5622 ablation model, and suggest that this pathology is not due to brain edema formation but to CP related pathology. Given the wide utilization of the iDTR allele and the Cx3cr1-iDTR model, it is crucial to fully characterize this pathology to understand the underlying causal mechanisms. Specifically, caution is needed when utilizing this model to interpret subtle neurologic functional changes that are thought to be mediated by microglia but could, instead, be due to CSF/ventricular loss in the genetic ablation model.

Diphtheria–tetanus–pertussis vaccine: past, current & future

The diphtheria–tetanus–pertussis (DTP) vaccine can prevent diphtheria, tetanus and pertussis. The component antigens of the DTP vaccine had long been monovalent vaccines. The pertussis vaccine was licensed in 1914. The same year, the mixtures of diphtheria toxin and antitoxin were put into use. In 1926, alum-precipitated diphtheria toxoid was registered, and in 1937 adsorbed tetanus toxoid was put on the market. The development of numerous effective DTP vaccines quickly stimulated efforts to combine DTP with other routine vaccines for infants. This overview covers the most important information regarding the invention of DTP vaccines, their modifications and the needs that should be focused on in the future.

Cutaneous infection by non-diphtheria-toxin producing and penicillin-resistant Corynebacterium diphtheriae strain in a patient with diabetes mellitus

Diphtheria is a potentially fatal infection, mostly caused by diphtheria toxin (DT)-producing Corynebacterium diphtheriae strains. During the last decades, the isolation of DT-producing C. diphtheriae strains has been decreasing worldwide. However, non-DT-producing C. diphtheriae strains emerged as causative agents of cutaneous and invasive infections. Although endemic in countries with warm climates, cutaneous diphtheria is rarely reported in Brazil. Presently, an unusual case of skin lesion in a Brazilian elderly diabetic patient infected by a penicillin-resistant non-DT-producing C. diphtheriae strain was reported. Laboratory diagnosis included mass spectrometry and multiplex PCR analyses. Since cutaneous diphtheria lesions are possible sources of secondary diphtheria cases and systemic diseases and considering that penicillin is the first line of antimicrobial agent for the treatment of these infections, the detection of penicillin-resistant strains of diphtheria bacilli should be a matter of concern. Thus, cases similar to the presently reported should be appropriately investigated and treated, particularly in patients with risk factor (s) for the development of C. diphtheriae invasive infections, such as diabetes. Moreover, health professionals must be aware of the presence of C. diphtheriae in cutaneous lesions of lower limbs, a common type of morbidity in diabetic patients, especially in tropical and subtropical countries.

A Pencil-Lead Immunosensor for the Rapid Electrochemical Measurement of Anti-Diphtheria Toxin Antibodies

Diphtheria is a vaccine-preventable disease, yet immunization can wane over time to non-protective levels. We have developed a low-cost, miniaturized electroanalytical biosensor to quantify anti-diphtheria toxin (DTx) immunoglobulin G (anti-DTx IgG) antibody to minimize the risk for localized outbreaks. Two epitopes specific to DTx and recognized by antibodies generated post-vaccination were selected to create a bi-epitope peptide, biEP, by synthesizing the epitopes in tandem. The biEP peptide was conjugated to the surface of a pencil-lead electrode (PLE) integrated into a portable electrode holder. Captured anti-DTx IgG was measured by square wave voltammetry from the generation of hydroquinone (HQ) from the resulting immunocomplex. The performance of the biEP reagent presented high selectivity and specificity for DTx. Under the optimized working conditions, a logarithmic calibration curve showed good linearity over the concentration range of 10−5–10−1 IU mL−1 and achieved a limit of detection of 5 × 10−6 IU mL−1. The final device proved suitable for interrogating the immunity level against DTx in actual serum samples. Results showed good agreement with those obtained from a commercial enzyme-linked immunosorbent assay. In addition, the flexibility for conjugating other capture molecules to PLEs suggests that this technology could be easily adapted to the diagnoses of other pathogens.

Diphtheria

Diphtheria is caused by toxin-producing bacteria, Corynebacterium diphtheriae, and less frequently by one of two other, zoonotic, Corynebacteria. Diphtheria toxin destroys tissue, which builds up in the throat and tonsils, making breathing and swallowing almost impossible. The bacteria are transmitted by respiratory droplets, by direct physical contact with skin lesions, via secretions from infected patients, or contaminated materials. Clinically, tonsillitis, pharyngitis, laryngitis, and skin infections (wound infection; ulcers) appear; diphtheria once was a terrible killer of young children. Antibiotics (penicillin, erythromycin, others) are used to eradicate the bacteria; for respiratory infections, diphtheria antitoxin is used to neutralize circulating toxins and reduce/prevent complications like myocarditis, neuritis (nerve palsies). Case fatality rates of up to 10% have been reported during diphtheria outbreaks, and are even higher in settings where diphtheria antitoxin is unavailable. Diphtheria vaccines consist of inactivated toxins, called toxoids, and are available in combinations with other antigens such as tetanus, pertussis, and others. These combinations are usually well-tolerated, local reactions are the most frequently observed side effects. Efficacy studies are not available but various observational studies consistently indicate high vaccine effectiveness between 87% and 96%.

Diphtheria Toxin Based Bivalent Anti-cMPL Immunotoxin Effectively Depletes Human Hematopoietic Stem and Progenitor Cells

Patient conditioning is a critical initial step in hematopoietic stem and progenitor cell (HSPC) transplantation procedures to enable marrow engraftment of infused cells. Preparative regimens have traditionally been achieved by delivering cytotoxic doses of chemotherapeutic agents, with or without radiation. However, these regimens impair host immune function and are associated with significant morbidity. The use of monoclonal antibodies, either alone or conjugated to an internalizing toxin, to target specific antigens on hematopoietic cells has been proposed as a tractable alternative, especially in contexts, such as ex vivo autologous gene therapy, where preservation of immunity is desired. Efficient clearance of marrow has been demonstrated in preclinical models using CD45- or CD117-targeting antibodies conjugated to the plant toxin Saporin. However, this approach still awaits demonstration of long-term safety and efficacy in humans. In this study, we investigated whether toxin-conjugated antibodies targeting the cMPL receptor on HSPCs can provide the basis for a conditioning regimen prior to transplant. Thrombopoietin (TPO) and its receptor cMPL act as primary regulators of HSPC self-renewal and survival. The TPO:cMPL axis also regulates megakaryopoiesis and platelet production but, unlike CD45 and CD117 proteins, cMPL is otherwise not expressed in other blood cell types or in non-hematopoietic tissues. Hence, this approach may uniquely allow effective and specific depletion of host HSCs while sparing most hematopoietic progenitors and mature blood cells. To investigate cMPL as an antigen for targeted depletion of human HSPCs, we produced a recombinant bivalent anti-cMPL single-chain variable fragment (sc(FV) 2) (Orita et al. Blood 2004) fused with diphtheria toxin truncated at residue 390 (DT390) to prevent toxin internalization in off-target cells. We first confirmed the cMPL receptor-dependent cytotoxic effects of the anti-cMPL-DT390 conjugate in a HEK293A cell line engineered to express the human cMPL receptor. We observed marked cellular killing in vitro (IC50 = 21 pM) compared to the cMPL-negative control HEK293A cell line (Fig. A). Next, we assessed anti-cMPL-DT390 for its ability to inhibit growth of human CD34+ cells in vitro. G-CSF mobilized peripheral blood (PB) CD34+ cells were obtained from five healthy individuals. Surface expression of cMPL was compared by flow cytometry in subsets increasingly enriched in cells with long-term repopulating activity, including bulk CD34+, CD34+CD38- and CD34+CD38-CD90+CD45RA-CD49f+ cells. Levels of cMPL expression increased congruently with levels of HSC purity (Fig. B). Consistent with a cMPL dependent cytotoxic effect, increased cellular death was measured in populations expressing higher densities of cMPL receptors (IC50 = 104 nM), suggesting preferential targeting of the most primitive hematopoietic compartment (Fig. C). We then assessed whether anti-cMPL-DT390 could safely target and deplete human HSPCs in vivo in humanized NBSGW immunodeficient mice. At 12 weeks post-transplantation, engrafted animals (mean 19.8% CD45+ cells in PB) received a single maximum tolerated dose of 1.2 mg/kg anti-cMPL-DT390 (n=7) or vehicle control solution (n=7) by tail vein injection. HSPC depletion was assayed by measuring human myeloid (CD45+CD13+) chimerism in the mouse PB after antibody administration. We observed a gradual decline in HSPC activity, as represented by the decreased production of human myeloid cells following administration of anti-cMPL-DT390, peaking at 6 weeks with a 2.6-fold reduction in frequency of human CD45+CD13+ cells compared to untreated animals (p = 0.003) (Fig. D). Overall, our study provides proof-of-concept that bivalent anti-cMPL immunotoxin can effectively target and deplete human HSPCs, and may thus provide a novel nontoxic preparative approach to improve HSPC engraftment in transplantation for genetic and other nonmalignant disorders. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Mature Osteoblasts Are Critical Bone Marrow Niche Cells Regulating Multiple Myeloma Cell Growth, at Least in Part, By Affecting the Local Immune Microenvironment

Despite high response rates to new therapies, most multiple myeloma (MM) patients relapse and become refractory to treatment. This is likely because MM cells enter dormancy and reactivate only under permissive conditions. Cellular dormancy is ubiquitous in normal tissues as well as in malignant, non-malignant, and metastasizing tumors. Dormant tumor cells (DTC) are protected from therapies which can reactivate later at the time of disease progression, resulting in relapsed refractory MM (RRMM). The tumor microenvironment (TME) is a key determinant of tumor dormancy and reactivation. We have previously shown that inhibition of Activin A in SCID mice transplanted with human MM.1S increased osteoblast numbers and resulted in inhibition of MM growth (Vallet et al., PNAS). We hypothesize that MM-DTCs reside in osteoblastic niche in the BM TME. Our long-term goal is to identify factors regulating DTCs in MM and develop strategies for long term disease control. We have generated mice in which mature osteoblasts can be postnatally deleted in an inducible and reversible manner. To achieve this, mice carrying floxed diphtheria toxin receptor (DTR) alleles were mated with mice expressing Cre-recombinase driven by the osteocalcin promoter (OC-Cre). This led to the expression of DTR in mature osteoblasts (OC-Cre/iDTR) only. The control mice were littermates lacking the OC-Cre allele (iDTR). Until the treatment with diphtheria toxin (DT), the OC-Cre/iDTR mice were immunocompetent compared to iDTR mice, as they did not show any differences in immune cell populations by flow cytometry, and did not show any skeletal phenotype, as measured by micro-computed CT (micro-CT). We chose to induce mature osteoblast-deficiency at 8-weeks of age to allow skeletal maturation which is assumed to be completed by 6-7weeks of age. To induce postnatal deletion of mature osteoblasts, the OC-Cre/iDTR and control mice both were treated with 50 µg/kg DT once a week, beginning at 8-weeks of age. Micro-CT analysis showed a significant increase in cortical porosity within 1-week after DT injection. 8-weeks of DT treatment significantly reduced trabecular bone fraction (BV/TV), trabecular numbers (Tb.N), and bone mineral density (BMD) with a significant increase in trabecular spacing (Tb.Sp). Immunohistochemistry for osteocalcin showed rapid loss of mature and endosteal osteoblasts (N.Ob/T.Ar). This was accompanied with a marked decreased in serum sclerostin and serum osteocalcin levels suggesting reduced osteocytes and mature osteoblasts, respectively. Importantly, serum CTX levels or numbers of osteoclasts (N.Oc/T.Ar) were unchanged. To study MM engraftment and progression, 3x10 6 5TGM1 luciferase tdTomato positive (5TGM1-Luc-Tom) MM cells were injected into the tibia of OC-Cre/iDTR and control iDTR mice followed by weekly injection of DT for 8-weeks. Flow cytometry analysis showed a 4-fold increase in the 5TGM1-Tom cells in the BM from OC-Cre/iDTR mice compared to controls. Bioluminescence imaging (BLI) for 5TGM1-Luc-Tom cells at 4-weeks showed dramatic increase in the OC-Cre/iDTR mice compared to controls. Interestingly, by 8-weeks, the BLI imaging showed 5TGM1-Luc-Tom cells in other long-bones of OC-Cre/iDTR mice but not the controls. To identify local BM TME changes, we assessed the levels of 200 cytokines and growth factors in the BM from these mice. The levels of immune regulatory cytokines, such as IL-17F and IL-21, were found to be decreased in the OC-Cre/iDTR mice compared to control iDTR mice, both after 4-weeks from intratibial 5TGM1 cell injection. This was accompanied by significant decrease in the fraction of CD4+ T-helper and Th17+ cells in the OC-Cre/iDTR mice compared to controls, as assessed by flow cytometry. These data show that MM cells engraft and proliferate rapidly within the BM in the absence of mature osteoblasts and have a propensity to migrate to other long bones with time. This may be partially mediated by the altered immune cell profile in the BM in the absence of mature osteoblasts. These data further suggest that expanding the mature osteoblast niche may provide novel therapeutic avenues and reduce disease burden creating an environment for long term tumor control. Importantly, this model will allow us to understand the osteoblastic niche for MM and the mechanisms of activation and dormancy, at least in part, via a role of the immune cells. Disclosures Fulzele: Constellation Pharma: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company. Raje: Celgene, Amgen, Bluebird Bio, Janssen, Caribou, and BMS: Other.

Generation of a Spiral Ganglion Neuron Degeneration Mouse Model

Spiral ganglion neurons (SGNs) can be injured by a wide variety of insults. However, there still is a lack of degeneration models to specifically damage the SGNs without disturbing other types of cells in the inner ear. This study aims to generate an SGN-specific damage model using the Cre-LoxP transgenic mouse strains. The Cre-inducible diphtheria toxin receptor (iDTR+/+) knock-in mouse strain was crossed with a mouse strain with Cre activity specific to neurons (NeflCreER/CreER). Expression of the Cre-recombinase activity was evaluated using the reporter mouse strain Ai9 at pre-hearing, hearing onset, and post-hearing stages. Accordingly, heterozygous NeflCreER/+;iDTR+/– mice were treated with tamoxifen on postnatal days 1–5 (P1–5), followed by diphtheria toxin (DT) or vehicle injection on P7, P14, and P21 to evaluate the SGN loss. Robust tamoxifen-induced Cre-mediated Ai9 tdTomato fluorescence was observed in the SGN area of heterozygous NeflCreER/+;Ai9+/– mice treated with tamoxifen, whereas vehicle-treated heterozygote mice did not show tdTomato fluorescence. Compared to vehicle-treated NeflCreER/+;iDTR+/– mice, DT-treated NeflCreER/+;iDTR+/– mice showed significant auditory brainstem response (ABR) threshold shifts and SGN cell loss. Hair cell count and functional study did not show significant changes. These results demonstrate that the NeflCreER/CreER mouse strain exhibits inducible SGN-specific Cre activity in the inner ear, which may serve as a valuable SGN damage model for regeneration research of the inner ear.

Significance of diphtheria toxin in neuropathy development and protective effect of nerves growth factor

In experiments in vitro and in vivo competitive relationships of diphtheria toxin and nerves growth factor were shown: in organotypical culture (300 sensor ganglions) and in model of experimental diphtheria neuropathy in 66 white rats. There were given evidences of protective action of nerves growth factor, inhibiting the toxic effect of diphtheria toxin. Contents of free toxin and specific immune comlexes was studied in 25 children with diphtheria polyneuropathy. Favourable outcome of disease was observed in children with high presence of nerves growth factor in blood serum, thus giving new approach in treatment of nervous system diseases.