HEMOPERFUSION USING THE LPS-SELECTIVE MESOPOROUS POLYMERIC ADSORBENT IN SEPTIC SHOCK: A MULTICENTER RANDOMIZED CLINICAL TRIAL.

ABSTRACT: Extracorporeal hemoperfusion (EHP) may improve the course and outcomes of patients with septic shock by targeting cytokines or bacterial endotoxins (lipopolysaccharide [LPS]). Here, we present the results of a multicenter randomized controlled trial ( clinicaltrials.gov/ct2/show/NCT04827407 ) to assess the efficiency and safety of Efferon LPS hemoperfusion cartridges engineered for multimodal targeting LPS, host-derived cytokine, and damage-associated molecule pattern molecules. Patients with intra-abdominal sepsis (IAS) and septic shock (Sepsis-3) were subjected to EHP procedures (n = 38). Control patients with IAS and septic shock (n = 20) were treated using conventional protocols without EHP. The primary end point was resolution of septic shock. Secondary end points included MAP, vasopressor drug dose, partial pressure of arterial oxygen/fraction of inspired oxygen ratio, Sequential Organ Failure Assessment score, length of stay in the intensive care unit, and satisfaction with device use by a 5-point Likert scale. Clinical laboratory tests for a blood cells count, lactate and creatinine concentration, nephelometry test for C-reactive protein, immunochemiluminescent test for procalcitonin, and immunoenzyme analysis for IL-6 concentration were used to monitor the EHP effect versus the control group. Data were analyzed followed the intention-to-treat approach. Wilcoxon STATA 16.0 (StataCorp, College Station, TX) and Excel 2019 with XLStat 2019 add-in (Addinsoft, Paris, France) were used for statistical analysis of the results. The Fine and Gray method of competing risks was used to analyze the primary end point and other data representing the time to event. EHP resulted in a significant and rapid increase in MAP and partial pressure arterial oxygen/fraction of inspired oxygen ratio, progressive decline in norepinephrine doses, and multiorgan deficiency, as evaluated by Sequential Organ Failure Assessment scores. Importantly, EHP led to significantly rapid cumulative mechanical ventilation weaning compared with the control group (subdistribution hazard ratio, 2.5; P = 0.037). Early 3-day mortality was significantly reduced in the Efferon LPS versus control group; however, no significant improvements in survival in 14 and 28 days were revealed. Laboratory tests showed rapidly decreased levels of LPS, procalcitonin, C-reactive protein, IL-6, creatinine, leukocytes, and neutrophils only in the Efferon LPS group. Results demonstrate that EHP with Efferon LPS is a safe procedure to abrogate septic shock and normalize clinical and pathogenically relevant biomarkers in patients with IAS.

Association of NEF2L2 Rs35652124 Polymorphism with Nrf2 Induction and Genotoxic Stress Biomarkers in Autism.

Increased oxidative/genotoxic stress is known to impact the pathophysiology of ASD (autism spectrum disorder). Clinical studies, however, reported limited, heterogeneous but promising responses to treatment with antioxidant remedies. We determined whether the functional polymorphism of the Nrf2 gene, master regulator of anti-oxidant adaptive reactions to genotoxic stress, links to the genotoxic stress responses and to an in vitro effect of a NRF2 inductor in ASD children. Oxidative stress biomarkers, adaptive responses to genotoxic/oxidative stress, levels of master antioxidant regulator Nrf2 and its active form pNrf2 before and after inducing by dimethyl fumarate (DMF), and promotor rs35652124 polymorphism of NFE2L2 gene encoding Nrf2 were studied in children with ASD (n = 179). Controls included healthy adults (n = 101). Adaptive responses to genotoxicity as indicated by H2AX and cytoprotection by NRF2 contents positively correlated in ASD children with a Spearman coefficient of R = 0.479 in T+, but not CC genotypes. ASD children with NRF2 rs35652124 CC genotype demonstrated significantly higher H2AX content (0.652 vs. 0.499 in T+) and pNrf2 induction by DMF, lowered 8-oxo-dG concentration in plasma and higher cfDNA/plasma nuclease activity ratio. Our pilot findings suggest that in ASD children the NEF2L2 rs35652124 polymorphism impacts adaptive responses that may potentially link to ASD severity. Our data warrant further studies to reveal the potential for NEF2L2 genotype-specific and age-dependent repurposing of DMF and/or other NRF2-inducing drugs.

Peroxynitrite in the tumor microenvironment changes the profile of antigens allowing escape from cancer immunotherapy.

Cancer immunotherapy often depends on recognition of peptide epitopes by cytotoxic T lymphocytes (CTLs). The tumor microenvironment (TME) is enriched for peroxynitrite (PNT), a potent oxidant produced by infiltrating myeloid cells and some tumor cells. We demonstrate that PNT alters the profile of MHC class I bound peptides presented on tumor cells. Only CTLs specific for PNT-resistant peptides have a strong antitumor effect in vivo, whereas CTLs specific for PNT-sensitive peptides are not effective. Therapeutic targeting of PNT in mice reduces resistance of tumor cells to CTLs. Melanoma patients with low PNT activity in their tumors demonstrate a better clinical response to immunotherapy than patients with high PNT activity. Our data suggest that intratumoral PNT activity should be considered for the design of neoantigen-based therapy and also may be an important immunotherapeutic target.

FIB-DIC Residual Stress Evaluation in Shot Peened VT6 Alloy Validated by X-ray Diffraction and Laser Speckle Interferometry.

Ga-ion micro-ring-core FIB-DIC evaluation of residual stresses in shot peened VT6 (Ti-6Al-4V) alloy was carried out and cross-validated against other non-destructive and semi-destructive residual stresses evaluation techniques, namely, the conventional sin2ψ X-ray diffraction and mechanical hole drilling. The Korsunsky FIB-DIC method of Ga-ion beam micro-ring-core milling within FIB-SEM with Digital Image Correlation (DIC) deformation analysis delivered spatial resolution down to a few micrometers, while the mechanical drilling of circular holes of ~2 mm diameter with laser speckle interferometry monitoring of strains gave a rough spatial resolution of a few millimeters. Good agreement was also found with the X-ray diffraction estimates of residual stress variation profiles as a function of depth. These results demonstrate that FIB-DIC provides rich information down to the micron scale, it also allows reliable estimation of macro-scale residual stresses.

Oxidized Cell-Free DNA Rapidly Skews the Transcriptional Profile of Brain Cells toward Boosting Neurogenesis and Neuroplasticity.

Cell-free DNA (cfDNA) is liberated and accumulated in neural tissue due to cell damage. The oxidative and nitrosative stress in the brain that accompanies various pathological conditions has been shown to increase the oxidation of cellular and cell-free DNA. Whether the high concentration of non-oxidized and oxidized cfDNA may affect the transcriptome response of brain cells has not been studied. In the current work, we studied whether cfDNA fragments affect several key pathways, including neurogenesis, at the level of gene expression in brain cells. In the study, primary rat cerebellum cell cultures were used to assess the effects of oxidized and non-oxidized cfDNA on the expression of 91 genes in brain cells. We found that only oxidized cfDNA, not non-oxidized cfDNA, significantly altered the transcription in brain cells in 3 h. The pattern of change included all 10 upregulated genes (S100A8, S100A9, S100b, TrkB, Ngf, Pink1, Aqp4, Nmdar, Kcnk2, Mapk1) belonging to genes associated with neurogenesis and neuroplasticity. The expression of inflammatory and apoptosis genes, which oppose neurogenesis, decreased. The results show that the oxidized form of cfDNA positively regulates early gene expression of neurogenesis and neuroplasticity. At the same time, the question of whether chronic elevation of cfDNA concentration alters brain cells remains unexplored.

Effect of Xenon Treatment on Gene Expression in Brain Tissue after Traumatic Brain Injury in Rats.

The overactivation of inflammatory pathways and/or a deficiency of neuroplasticity may result in the delayed recovery of neural function in traumatic brain injury (TBI). A promising approach to protecting the brain tissue in TBI is xenon (Xe) treatment. However, xenon's mechanisms of action remain poorly clarified. In this study, the early-onset expression of 91 target genes was investigated in the damaged and in the contralateral brain areas (sensorimotor cortex region) 6 and 24 h after injury in a TBI rat model. The expression of genes involved in inflammation, oxidation, antioxidation, neurogenesis and neuroplasticity, apoptosis, DNA repair, autophagy, and mitophagy was assessed. The animals inhaled a gas mixture containing xenon and oxygen (ϕXe = 70%; ϕO2 25-30% 60 min) 15-30 min after TBI. The data showed that, in the contralateral area, xenon treatment induced the expression of stress genes (Irf1, Hmox1, S100A8, and S100A9). In the damaged area, a trend towards lower expression of the inflammatory gene Irf1 was observed. Thus, our results suggest that xenon exerts a mild stressor effect in healthy brain tissue and has a tendency to decrease the inflammation following damage, which might contribute to reducing the damage and activating the early compensatory processes in the brain post-TBI.

Oxidized cell-free DNA as a stress-signaling factor activating the chronic inflammatory process in patients with autism spectrum disorders.

BACKGROUND: Autism spectrum disorders (ASD) are known to be associated with an inflammatory process related to immune system dysfunction. This study's aim was to investigate the role of cell-free DNA in chronic inflammatory process in ASD patients. METHODS: The study included 133 ASD patients and 27 healthy controls. Sixty-two ASD patients were demonstrated to have mild-to-moderate disease severity (group I) and 71 individuals to have severe ASD (group II). Plasma cell-free (cf) DNA characteristics, plasma cytokine concentrations, expression of the genes for NFкB1 transcription factor and pro-inflammatory cytokines TNFα, IL-1ß and IL-8 in peripheral blood lymphocytes (PBL) of ASD patients, and unaffected controls were investigated. Additionally, in vitro experiments with oxidized DNA supplementation to PBL cultures derived from ASD patients and healthy controls were performed. RESULTS: The data indicates that ASD patients have demonstrated increased cfDNA concentration in their circulation. cfDNA of patients with severe ASD has been characterized by a high abundance of oxidative modification. Furthermore, ASD patients of both groups have shown elevated plasma cytokine (IL-1ß, IL-8, IL-17A) levels and heightened expression of genes for NFкB1 nuclear factor and pro-inflammatory cytokines TNFα, IL-1ß, and IL-8 in PBL. In vitro experiments have shown that NF-κB/cytokine mRNA expression profiles of ASD patient PBL treated with oxidized DNA fragments were significantly different from those of healthy controls. CONCLUSIONS: It may be proposed that oxidized cfDNA plays a role of stress-signaling factor activating the chronic inflammatory process in patients with ASD.

Changes of KEAP1/NRF2 and IKB/NF-κB Expression Levels Induced by Cell-Free DNA in Different Cell Types.

Cell-free DNA (cfDNA) is a circulating DNA of nuclear and mitochondrial origin mainly derived from dying cells. Recent studies have shown that cfDNA is a stress signaling DAMP (damage-associated molecular pattern) molecule. We report here that the expression profiles of cfDNA-induced factors NRF2 and NF-κB are distinct depending on the target cell's type and the GC-content and oxidation rate of the cfDNA. Stem cells (MSC) have shown higher expression of NRF2 without inflammation in response to cfDNA. In contrast, inflammatory response launched by NF-κB was dominant in differentiated cells HUVEC, MCF7, and fibroblasts, with a possibility of transition to massive apoptosis. In each cell type examined, the response for oxidized cfDNA was more acute with higher peak intensity and faster resolution than that for nonoxidized cfDNA. GC-rich nonoxidized cfDNA evoked a weaker and prolonged response with proinflammatory component (NF-κB) as predominant. The exploration of apoptosis rates after adding cfDNA showed that cfDNA with moderately increased GC-content and lightly oxidized DNA promoted cell survival in a hormetic manner. Novel potential therapeutic approaches are proposed, which depend on the current cfDNA content: either preconditioning with low doses of cfDNA before a planned adverse impact or eliminating (binding, etc.) cfDNA when its content has already become high.

Modeling and simulation of a low-grade urinary bladder carcinoma.

In this work, we present a mathematical model of the initiation and progression of a low-grade urinary bladder carcinoma. We simulate the crucial processes affecting tumor growth, such as oxygen diffusion, carcinogen penetration, and angiogenesis, within the framework of the urothelial cell dynamics. The cell dynamics are modeled using the discrete technique of cellular automata, while the continuous processes of carcinogen penetration and oxygen diffusion are described by nonlinear diffusion-absorption equations. As the availability of oxygen is necessary for tumor progression, processes of oxygen transport to the tumor growth site seem most important. Our model yields a theoretical insight into the main stages of development and growth of urinary bladder carcinoma with emphasis on the two most common types: bladder polyps and carcinoma in situ. Analysis of histological structure of bladder tumor is important to avoid misdiagnosis and wrong treatment. We expect our model to be a valuable tool in the study of bladder cancer progression due to the exposure to carcinogens and the oxygen dependent expression of genes promoting tumor growth. Our numerical simulations have good qualitative agreement with in vivo results reported in the corresponding medical literature.

Cellular immunotherapy study of prostate cancer patients and resulting IgG responses to peptide epitopes predicted from prostate tumor-associated autoantigens.

The immunogenicity of a cellular immunotherapy using genetically modified vaccines to express α(1,3)galactosyl epitopes (αGal) was evaluated in advanced prostate cancer (PC) patients. In this dose escalation phase I study, we report safety, feasibility, and immunologic data of an immunotherapy composed of 2 human PC cell lines engineered to express αGal epitopes (HyperAcute-Prostate, HAP, NewLink Genetics). Eight patients received up to 12 biweekly vaccinations with HAP. Enrolled patients (aged range, 53-85 y) had American Joint Committee on Cancer stage IV, any T, any N, M1, Eastern Cooperative Oncology Group PS≤2, at least 1 prior hormonal treatment and <3 prior chemotherapies, adequate bone marrow and organ function, and albumin ≥3.0 g/dL. Serum IgG antibodies to synthetic peptides overexpressed in PC were determined by enzyme-linked immunosorbent assay. Results indicate that HAP immunotherapy induced humoral immune responses to autoantigens in 2 of 8 patients. These patients developed IgG antibody to multiple epitopes overexpressed in PC after immunization. These responding patients received higher doses of the immunotherapy suggesting a dose response. Two immunogenic proteins (prostate-specific membrane antigen, hepsin) belong to the extracellular molecules family participating in malignant cell invasion. Median overall survival for patients was 25.1 months with 1 patient surviving over 70 months with stable PSA and bone metastasis before expiring of other causes. Three of 8 patients showed PSA stabilization (>100 d). In conclusion, HAP immunotherapy induces IgG responses to epitopes from autoantigens overexpressed in PC suggesting dose-dependent effect. HAP represents a viable immunotherapy approach to induce immune responses against tumor cells and may provide clinical benefit with minimal toxicity.

Tumor-infiltrating myeloid cells induce tumor cell resistance to cytotoxic T cells in mice.

Cancer immunotherapeutic approaches induce tumor-specific immune responses, in particular CTL responses, in many patients treated. However, such approaches are clinically beneficial to only a few patients. We set out to investigate one possible explanation for the failure of CTLs to eliminate tumors, specifically, the concept that this failure is not dependent on inhibition of T cell function. In a previous study, we found that in mice, myeloid-derived suppressor cells (MDSCs) are a source of the free radical peroxynitrite (PNT). Here, we show that pre-treatment of mouse and human tumor cells with PNT or with MDSCs inhibits binding of processed peptides to tumor cell-associated MHC, and as a result, tumor cells become resistant to antigen-specific CTLs. This effect was abrogated in MDSCs treated with a PNT inhibitor. In a mouse model of tumor-associated inflammation in which the antitumor effects of antigen-specific CTLs are eradicated by expression of IL-1ß in the tumor cells, we determined that therapeutic failure was not caused by more profound suppression of CTLs by IL-1ß-expressing tumors than tumors not expressing this proinflammatory cytokine. Rather, therapeutic failure was a result of the presence of PNT. Clinical relevance for these data was suggested by the observation that myeloid cells were the predominant source of PNT in human lung, pancreatic, and breast cancer samples. Our data therefore suggest what we believe to be a novel mechanism of MDSC-mediated tumor cell resistance to CTLs.

Udp-glucose dehydrogenase as a novel field-specific candidate biomarker of prostate cancer.

Uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) catalyzes the oxidation of UDP-glucose to yield UDP-glucuronic acid, a precursor for synthesis of glycosaminoglycans and proteoglycans that promote aggressive prostate cancer (PC) progression. The purpose of our study was to determine if the UGDH expression in normal appearing acini (NAA) from cancerous glands is a candidate biomarker for PC field disease/effect assayed by quantitative fluorescence imaging analysis (QFIA). A polyclonal antibody to UGDH was titrated to saturation binding and fluorescent microscopic images acquired from fixed, paraffin-embedded tissue slices were quantitatively analyzed. Specificity of the assay was confirmed by Western blot analysis and competitive inhibition of tissue labeling with the recombinant UGDH. Reproducibility of the UGDH measurements was high within and across analytical runs. Quantification of UGDH by QFIA and Reverse-Phase Protein Array analysis were strongly correlated (r = 0.97), validating the QFIA measurements. Analysis of cancerous acini (CA) and NAA from PC patients vs. normal acini (NA) from noncancerous controls (32 matched pairs) revealed significant (p < 0.01) differences, with CA (increased) vs. NA, NAA (decreased) vs. NA and CA (increased) vs. NAA. Areas under the Receiver Operating Characteristic curves were 0.68 (95% CI: 0.59-0.83) for NAA and 0.71 (95% CI: 0.59-0.83) for CA (both vs. NA). These results support the UGDH content in prostatic acini as a novel candidate biomarker that may complement the development of a multi-biomarker panel for detecting PC within the tumor adjacent field on a histologically normal biopsy specimen.

The effect of the nonionic block copolymer pluronic P85 on gene expression in mouse muscle and antigen-presenting cells.

DNA vaccines can be greatly improved by polymer agents that simultaneously increase transgene expression and activate immunity. We describe here Pluronic P85 (P85), a triblock copolymer of ethylene oxide (EO) and propylene oxide (PO) EO(26)-PO(40)-EO(26). Using a mouse model we demonstrate that co-administration of a bacterial plasmid DNA with P85 in a skeletal muscle greatly increases gene expression in the injection site and distant organs, especially the draining lymph nodes and spleen. The reporter expression colocalizes with the specific markers of myocytes and keratinocytes in the muscle, as well as dendritic cells (DCs) and macrophages in the muscle, lymph nodes and spleen. Furthermore, DNA/P85 and P85 alone increase the systemic expansion of CD11c+ (DC), and local expansion of CD11c+, CD14+ (macrophages) and CD49b+ (natural killer) cell populations. DNA/P85 (but not P85) also increases maturation of local DC (CD11c+ CD86+, CD11c+ CD80 +, and CD11c+ CD40+. We suggest that DNA/P85 promotes the activation and recruitment of the antigen-presenting cells, which further incorporate, express and carry the transgene to the immune system organs.

Identification of O-glycosylated decapeptides within the MUC1 repeat domain as potential MHC class I (A2) binding epitopes.

The MUC1 glycoprotein is considered a tumor antigen due to its over expression and aberrant glycosylation in cancer tissues. The latter results in appearance of new antigenic tumor specific glycopeptides not found on normal glycoforms of the mucin. MUC1 glycopeptides can be presented by APCs on MHC class II molecules to activate glycopeptide specific helper T-cells. No study has yet reported presentation of MUC1 glycopeptides on MHC class I molecules as stimulators of cytotoxic T-cells. In this study we show that human immunoproteasomes and cathepsin-L can generate octa to undecameric glycopeptides from the MUC1 repeat domain in vitro. We identified glycosylated fragments of which the decameric glycopeptide SAP10 [SAPDT(GalNAc)RPAPG] containing a single sugar binds with comparable strength to the MHC class I allele HLA A*0201 as predicted high-score binding epitopes of the tandem repeat. The same sequence glycosylated with the disaccharide Gal-GalNAc does not bind. The glycan on SAP10 is predicted by molecular modeling to either protrude out or point into the MHC groove. SAPDTRPAPG peptide and the respective glycopeptide stimulated cytotoxic T-cells in vitro. Our findings suggest that MUC1 tandem repeat glycopeptides are capable of activating both helper and cytotoxic T-cells and thus represent good candidates for further development as vaccines.

Nitrated alpha-synuclein immunity accelerates degeneration of nigral dopaminergic neurons.

BACKGROUND: The neuropathology of Parkinson's disease (PD) includes loss of dopaminergic neurons in the substantia nigra, nitrated alpha-synuclein (N-alpha-Syn) enriched intraneuronal inclusions or Lewy bodies and neuroinflammation. While the contribution of innate microglial inflammatory activities to disease are known, evidence for how adaptive immune mechanisms may affect the course of PD remains obscure. We reasoned that PD-associated oxidative protein modifications create novel antigenic epitopes capable of peripheral adaptive T cell responses that could affect nigrostriatal degeneration. METHODS AND FINDINGS: Nitrotyrosine (NT)-modified alpha-Syn was detected readily in cervical lymph nodes (CLN) from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxicated mice. Antigen-presenting cells within the CLN showed increased surface expression of major histocompatibility complex class II, initiating the molecular machinery necessary for efficient antigen presentation. MPTP-treated mice produced antibodies to native and nitrated alpha-Syn. Mice immunized with the NT-modified C-terminal tail fragment of alpha-Syn, but not native protein, generated robust T cell proliferative and pro-inflammatory secretory responses specific only for the modified antigen. T cells generated against the nitrated epitope do not respond to the unmodified protein. Mice deficient in T and B lymphocytes were resistant to MPTP-induced neurodegeneration. Transfer of T cells from mice immunized with N-alpha-Syn led to a robust neuroinflammatory response with accelerated dopaminergic cell loss. CONCLUSIONS: These data show that NT modifications within alpha-Syn, can bypass or break immunological tolerance and activate peripheral leukocytes in draining lymphoid tissue. A novel mechanism for disease is made in that NT modifications in alpha-Syn induce adaptive immune responses that exacerbate PD pathobiology. These results have implications for both the pathogenesis and treatment of this disabling neurodegenerative disease.

Altered recognition of antigen is a mechanism of CD8+ T cell tolerance in cancer.

Antigen-specific CD8+ T-cell tolerance, induced by myeloid-derived suppressor cells (MDSCs), is one of the main mechanisms of tumor escape. Using in vivo models, we show here that MDSCs directly disrupt the binding of specific peptide-major histocompatibility complex (pMHC) dimers to CD8-expressing T cells through nitration of tyrosines in a T-cell receptor (TCR)-CD8 complex. This process makes CD8-expressing T cells unable to bind pMHC and to respond to the specific peptide, although they retain their ability to respond to nonspecific stimulation. Nitration of TCR-CD8 is induced by MDSCs through hyperproduction of reactive oxygen species and peroxynitrite during direct cell-cell contact. Molecular modeling suggests specific sites of nitration that might affect the conformational flexibility of TCR-CD8 and its interaction with pMHC. These data identify a previously unknown mechanism of T-cell tolerance in cancer that is also pertinent to many pathological conditions associated with accumulation of MDSCs.

Dendritic cell-based full-length survivin vaccine in treatment of experimental tumors.

Survivin is a good candidate for cancer immunotherapy since it is overexpressed in most common human cancers, poorly expressed in most normal adult tissues and is essential for cancer cell survival. Previously, we and others have demonstrated that survivin-specific immune responses can be generated in mice and cancer patients. These responses resulted in a substantial antitumor effect. However, the fact that survivin is expressed in normal hematopoietic progenitor cells and endothelial cells may potentially limit the use of vaccination against survivin in the clinic due to possible toxicity. In this study, we have evaluated this risk by using dendritic cells (DC) transduced with an adenovirus encoding mutant human survivin (Ad-surv DCs). Immunization of mice with Ad-surv DCs resulted in generation of CD8 T cells recognizing multiple epitopes from mouse survivin. These responses provided significant antitumor effect against 3 different tumors EL-4 lymphoma, MC-38 carcinoma, and MethA sarcoma. Survivin-specific T-cells did not affect bone marrow hematopoietic progenitor cells and no autoimmune abnormalities were observed. However, as was the case with other tumor vaccines it provided only partial antitumor effect against established tumors. The existing paradigm suggests that generation of immune response against multiple tumor-associated antigens may provide a better antitumor effect. Here, we directly tested this hypothesis by combining vaccines targeting different tumor-associated proteins: survivin and p53. Despite the fact that combination of 2 vaccines generated potent antigen specific T-cell responses against both molecules they did not result in the improvement of antitumor effect in any of the tested experimental tumor models.

Hematopoietic progenitor cell mobilization in mice by sustained delivery of granulocyte colony-stimulating factor.

The objective of these studies was to determine the effect of sustained delivery of growth factors (GFs) on hematopoietic progenitor cells (HPCs) in mice. In these studies, granulocyte colony-stimulating factor (G-CSF) was administered using the poloxamer-based matrix, ProGelz (PG) and G-CSF, and pharmacokinetics (PKs) and HPC mobilization was assessed. A single injection of G-CSF formulated in PG (17% poloxamer-407 and 5% hydroxypropyl methylcellulose [HPMC]) administered to BALB/c mice mobilized HPC significantly more rapidly to the spleen, but not the blood, than multiple injections of saline-formulated G-CSF. Two days after a single injection of PG G-CSF, the frequency of colony-forming unit-culture (CFU-c) in the spleen was increased 289-fold compared with an 8-fold increase after 2 days of twice-daily injections of saline-formulated G-CSF. Indeed, 4 days of twice-daily G-CSF injections were required to achieve the same level of HPC mobilization. In contrast, a similar mobilization of HPC to the blood was observed between PG and saline-formulated G-CSF. The mechanism for the accelerated and increased mobilization to the spleen by the PG-formulation of G-CSF is due, in part, to its increased bioavailability (>1.5-fold), T(max) (6-fold), and prolonged elimination (Tbeta) half-life (>3-fold) as compared with a saline formulation. In addition, we observed a more rapid trafficking of the PG G-CSF to the marrow, which could also facilitate mobilization.

Sequence-variant repeats of MUC1 show higher conformational flexibility, are less densely O-glycosylated and induce differential B lymphocyte responses.

The human epithelial cancer mucin MUC1 is able to break tolerance and to induce humoral immune responses in healthy subjects and in cancer patients. We recently showed that clusters of sequence-variant repeats are interspersed in the repeat domain of MUC1 at high frequency, which should contribute to the structural and immunological features of the mucin. Here we elucidated the potential effects exerted by sequence-variant repeats on their O-glycosylation. Evidence from in vitro glycosylation with polypeptide N-acetylgalactosaminyltransferases GalNAc-T1 and GalNAc-T2 in concert with mass spectrometric analyses of in vivo glycosylated MUC1 probes from transiently transfected HEK293 cells indicated reduced glycosylation densities of repeats with three concerted replacements: AHGVTSAPESRPAPGSTAPA. The Pro to Ala replacement in STAPA exerts not only proximal effects on the ppGalNAc-T2 preferred site at -3 and -4, but also more distant effects on the ppGalNAc-T1 preferred site at -15 (TSAPESRPAPGSTAPA). We also examined the conformational changes of MUC1 glycopeptides induced by the concerted DT to ES replacements and revealed a higher conformational flexibility of ES/P peptides compared to DT/P peptides. Differences in conformational flexibilities and in O-glycosylation densities could underlie the observed differential humoral responses in humans. We were able to show that the natural immunoglobulin G (IgG) responses to the repeat domain of MUC1 in sera from nonmalignant control subjects are preferentially directed to variant repeat clusters. In contrast, the IgG response in patients with adenocarcinoma shifted to higher frequencies of preferential DTR peptide binding.