Severe intraventricular hemorrhage causes long-lasting structural damage in a preterm rabbit pup model.

BACKGROUND: Intraventricular hemorrhage causes significant lifelong mortality and morbidity, especially in preterm born infants. Progress in finding an effective therapy is stymied by a lack of preterm animal models with long-term follow-up. This study addresses this unmet need, using an established model of preterm rabbit IVH and analyzing outcomes out to 1 month of age. METHODS: Rabbit pups were delivered preterm and administered intraperitoneal injection of glycerol at 3 h of life and approximately 58% developed IVH. Neurobehavioral assessment was performed at 1 month of age followed by immunohistochemical labeling of epitopes for neurons, synapses, myelination, and interneurons, analyzed by means of digital quantitation and assessed via two-way ANOVA or Student's t test. RESULTS: IVH pups had globally reduced myelin content, an aberrant cortical myelination microstructure, and thinner upper cortical layers (I-III). We also observed a lower number of parvalbumin (PV)-positive interneurons in deeper cortical layers (IV-VI) in IVH animals and reduced numbers of neurons, synapses, and microglia. However, there were no discernable changes in behaviors. CONCLUSIONS: We have established in this preterm pup model that long-term changes after IVH include significant wide-ranging alterations to cortical organization and microstructure. Further work to improve the sensitivity of neurocognitive testing in this species at this age may be required. IMPACT: This study uses an established animal model of preterm birth, in which the rabbit pups are truly born preterm, with reduced organ maturation and deprivation of maternally supplied trophic factors. This is the first study in preterm rabbits that explores the impacts of severe intraventricular hemorrhage beyond 14 days, out to 1 month of age. Our finding of persisting but subtle global changes including brain white and gray matter will have impact on our understanding of the best path for therapy design and interventions.

Insulin-Like Growth Factor 1 in the Preterm Rabbit Pup: Characterization of Cerebrovascular Maturation following Administration of Recombinant Human Insulin-Like Growth Factor 1/Insulin-Like Growth Factor 1-Binding Protein 3.

Following preterm birth, serum levels of insulin-like growth factor 1 (IGF-1) decrease compared to corresponding in utero levels. A recent clinical trial indicated that supplementation with recombinant human (rh) IGF-1/rhIGF-binding protein 3 (rhIGF-1/rhIGFBP-3) prevents severe intraventricular hemorrhage (IVH) in extremely preterm infants. In a preterm rabbit pup model, we characterized endogenous serum and hepatic IGF-1, along with brain distribution of IGF-1 and IGF-1 receptor (IGF1R). We then evaluated the effects of rhIGF-1/rhIGFBP-3 on gene expression of regulators of cerebrovascular maturation and structure. Similar to preterm infants, serum IGF-1 concentrations decreased rapidly after preterm birth in the rabbit pup. Administration of rhIGF-1/rhIGFBP-3 restored in utero serum levels but was rapidly eliminated. Immunolabeled IGF1R was widely distributed in multiple brain regions, displaying an abundant density in the choroid plexus and sub-ependymal germinal zones. Increased IGF-1 immunoreactivity, distributed as IGF1R, was detected 4 h after rhIGF-1/rhIGFBP-3 administration. The rhIGF-1/rhIGFBP-3 treatment led to upregulation of choroid plexus genes involved in vascular maturation and structure, with corresponding protein translation for most of these genes. The preterm rabbit pup model is well suited for evaluation of IGF-1-based prevention of IVH. Administration of rhIGF-1/rhIGFBP-3 affects cerebrovascular maturation, suggesting a role for it in preventing preterm IVH.

Cell-free oxidized hemoglobin drives reactive oxygen species production and pro-inflammation in an immature primary rat mixed glial cell culture.

BACKGROUND: Germinal matrix intraventricular hemorrhage (GM-IVH) is associated with deposition of redox active cell-free hemoglobin (Hb), derived from hemorrhagic cerebrospinal fluid (CSF), in the cerebrum and cerebellum. In a recent study, using a preterm rabbit pup model of IVH, intraventricularly administered haptoglobin (Hp), a cell-free Hb scavenger, partially reversed the damaging effects observed following IVH. Together, this suggests that cell-free Hb is central in the pathophysiology of the injury to the immature brain following GM-IVH. An increased understanding of the causal pathways and metabolites involved in eliciting the damaging response following hemorrhage is essential for the continued development and implementation of neuroprotective treatments of GM-IVH in preterm infant. METHODS: We exposed immature primary rat mixed glial cells to hemorrhagic CSF obtained from preterm human infants with IVH (containing a mixture of Hb-metabolites) or to a range of pure Hb-metabolites, incl. oxidized Hb (mainly metHb with iron in Fe3+), oxyHb (mainly Fe2+), or low equivalents of heme, with or without co-administration with human Hp (a mixture of isotype 2-2/2-1). Following exposure, cellular response, reactive oxygen species (ROS) generation, secretion and expression of pro-inflammatory cytokines and oxidative markers were evaluated. RESULTS: Exposure of the glial cells to hemorrhagic CSF as well as oxidized Hb, but not oxyHb, resulted in a significantly increased rate of ROS production that positively correlated with the rate of production of pro-inflammatory and oxidative markers. Congruently, exposure to oxidized Hb caused a disintegration of the polygonal cytoskeletal structure of the glial cells in addition to upregulation of F-actin proteins in microglial cells. Co-administration of Hp partially reversed the damaging response of hemorrhagic CSF and oxidized Hb. CONCLUSION: Exposure of mixed glial cells to oxidized Hb initiates a pro-inflammatory and oxidative response with cytoskeletal disintegration. Early administration of Hp, aiming to minimize the spontaneous autoxidation of cell-free oxyHb and liberation of heme, may provide a therapeutic benefit in preterm infant with GM-IVH.

Long-term neurological effects of neonatal caffeine treatment in a rabbit model of preterm birth.

BACKGROUND: Neonatal caffeine treatment might affect brain development. Long-term studies show conflicting results on brain-related outcomes. Herein we aimed to investigate the long-term effects of neonatal caffeine administration in a rabbit model of preterm birth. METHODS: Preterm (born day 29) and term (day 32) pups were raised by wet nurses and allocated to treatment with saline or caffeine for 7 or 17 days. At pre-puberty, neurobehavioral tests were performed and brains were harvested for immunostaining of neurons, synapses, myelin, and astrocytes. RESULTS: Survival was lower in preterm saline pups than in controls, whereas caffeine-treated preterm pups did not differ from term control pups. Preterm saline pups covered less distance compared to controls and were more likely to stay in the peripheral zone of the open field. Corresponding differences were not seen in preterm caffeine pups. Preterm animals had lower neuron density compared to controls, which was not influenced by caffeine treatment. Synaptic density, astrocytes, and myelin were not different between groups. CONCLUSION: Caffeine appeared to be safe. All preterm rabbits had lower neuron density but anxious behavior seen in preterm saline rabbits was not seen in caffeine-treated preterm pups.

Visualisation of H2O2 penetration through skin indicates importance to develop pathway-specific epidermal sensing.

Elevated amounts of reactive oxygen species (ROS) including hydrogen peroxide (H2O2) are observed in the epidermis in different skin disorders. Thus, epidermal sensing of H2O2 should be useful to monitor the progression of skin pathologies. We have evaluated epidermal sensing of H2O2 in vitro, by visualising H2O2 permeation through the skin. Skin membranes were mounted in Franz cells, and a suspension of Prussian white microparticles was deposited on the stratum corneum face of the skin. Upon H2O2 permeation, Prussian white was oxidised to Prussian blue, resulting in a pattern of blue dots. Comparison of skin surface images with the dot patterns revealed that about 74% of the blue dots were associated with hair shafts. The degree of the Prussian white to Prussian blue conversion strongly correlated with the reciprocal resistance of the skin membranes. Together, the results demonstrate that hair follicles are the major pathways of H2O2 transdermal penetration. The study recommends that the development of H2O2 monitoring on skin should aim for pathway-specific epidermal sensing, allowing micrometre resolution to detect and quantify this ROS biomarker at hair follicles.Graphical abstract.

The heme and radical scavenger α1-microglobulin (A1M) confers early protection of the immature brain following preterm intraventricular hemorrhage.

BACKGROUND: Germinal matrix intraventricular hemorrhage (GM-IVH) is associated with cerebro-cerebellar damage in very preterm infants, leading to neurodevelopmental impairment. Penetration, from the intraventricular space, of extravasated red blood cells and extracellular hemoglobin (Hb), to the periventricular parenchyma and the cerebellum has been shown to be causal in the development of brain injury following GM-IVH. Furthermore, the damage has been described to be associated with the cytotoxic nature of extracellular Hb-metabolites. To date, there is no therapy available to prevent infants from developing either hydrocephalus or serious neurological disability. Mechanisms previously described to cause brain damage following GM-IVH, i.e., oxidative stress and Hb-metabolite toxicity, suggest that the free radical and heme scavenger α1-microglobulin (A1M) may constitute a potential neuroprotective intervention. METHODS: Using a preterm rabbit pup model of IVH, where IVH was induced shortly after birth in pups delivered by cesarean section at E29 (3 days prior to term), we investigated the brain distribution of recombinant A1M (rA1M) following intracerebroventricular (i.c.v.) administration at 24 h post-IVH induction. Further, short-term functional protection of i.c.v.-administered human A1M (hA1M) following IVH in the preterm rabbit pup model was evaluated. RESULTS: Following i.c.v. administration, rA1M was distributed in periventricular white matter regions, throughout the fore- and midbrain and extending to the cerebellum. The regional distribution of rA1M was accompanied by a high co-existence of positive staining for extracellular Hb. Administration of i.c.v.-injected hA1M was associated with decreased structural tissue and mitochondrial damage and with reduced mRNA expression for proinflammatory and inflammatory signaling-related genes induced by IVH in periventricular brain tissue. CONCLUSIONS: The results of this study indicate that rA1M/hA1M is a potential candidate for neuroprotective treatment following preterm IVH.

Stellate cells and mesenchymal stem cells in benign mammary stroma are associated with risk factors for breast cancer - an observational study.

BACKGROUND: It is not known whether stromal cells in benign breast tissue can mediate risk of breast cancer. We recently described aldehyde dehydrogenase 1 A1 (ALDH1) positive (+) cells in morphologically normal breast stroma of premenopausal women, and the data indicated that their distribution is associated with clinical risk factors for breast cancer. The aim of the present study was to define the identities of these cells using histologic and immunohistologic methods, and to investigate associations between those cells and hormonal and genetic risk factors in pre- and postmenopausal women. METHODS: Stroma of morphologically normal tissue was analyzed in samples from 101 well-characterized women whose breasts had been operated. Morphology and immunolabeling were applied to determine cell identities based on the putative stem cell markers ALDH1 and stage-specific embryonic antigen-3 (SSEA3), and immunophenotypes indicating mast cells or stellate cells. The results were compared with the patients' risk factors using regression analysis (two-tailed). RESULTS: ALDH1+ round/oval cells were associated with low parity in BRCA1/2 carriers (p = 0.022), while in non-BRCA1/2-carriers they were negatively associated with nulliparity (p = 0.057). In premenopausal women ALDH1+ round/oval cells were associated with family history (p = 0.058). SSEA3+ round/oval cells were morphologically and immunohistologically consistent with multilineage stress-enduring (Muse) cells, and these cells were independently associated with the breast cancer risk factors low parity (p = 0.015), family history (p = 0.021), and hormone use after menopause (p = 0.032). ALDH1+ spindle-shaped/polygonal cells were immunohistologically consistent with stellate cells, and were negatively associated with family history of breast cancer (p = 0.001). CONCLUSION: This study identified novel stromal cell types in benign breast tissue that have a potential for stratifying women for breast cancer risk.

Women with familial risk for breast cancer have an increased frequency of aldehyde dehydrogenase expressing cells in breast ductules.

BACKGROUND: Knowledge is limited regarding the association between stem cells in histologically benign breast tissue and risk factors for breast cancer, and hence we addressed this issue in the present study. Recently, we assessed the histology of benign breast tissue from cancer and non-cancer patients for cells positive for the putative stem cell marker aldehyde dehydrogenase 1 A1 (ALDH), and the findings indicated an association between expression of ALDH and the hormonal factors menopause and hormone therapy. The current investigation examined possible associations between various known clinical and genetic risk factors for breast cancer and cellular expression of ALDH in ductules in benign human breast tissue. METHODS: The study included breast surgery patients that were BRCA1/2 mutation carriers without breast cancer (n = 23), had BRCA1/2 (n = 28) or sporadic (n = 21) breast cancer, or required non-cancer-related mammoplasty (n = 34). The distribution and frequency of ALDH-immunolabelled cells were correlated to patient subgroups with different risk factors, using mammoplasty patients as a control group. Statistical analyses comprised linear and logistic regression, Spearman's rank test, Pearson's test, and Fisher's exact test. In two-tailed tests, p < 0.05 was considered significant. RESULTS: A strong association was found between family history of breast cancer and a high frequency of ALDH+ cells (p = 0.001) at all ductular levels in all groups, regardless of BRCA status, age, parity, or occurrence of cancer. In pre-menopausal non-BRCA cancer patients, the frequency of ALDH+ cells increased with age (p < 0.01) but decreased with increasing parity (p < 0.03). High frequencies of ALDH+ cells were found in the non-basal ductular levels in BRCA1 mutation carriers (p = 0.03), but in the basal ductular level in BRCA2 cancer patients (p = 0.02). Among post-menopausal patients, only on-going hormone replacement therapy was correlated with a high number of ALDH+ cells (p < 0.03). CONCLUSION: In histologically normal breast tissue, we found a positive association between the frequency of ductular ALDH+ cells and several breast cancer risk factors, particularly family history of this disease, which supports previous evidence that ALDH plays a role in breast cancer.

Evaluation of enhanced permeability effect and different linear energy transfer of radionuclides in a prostate cancer xenograft model.

We have previously investigated the biodistribution and therapy effect of a humanized monoclonal antibody targeting free prostate-specific antigen (fPSA) intended for theranostics of hormone-refractory prostate cancer. In the present study, we evaluated the off-target effect and different linear energy transfer (LET) radionuclides without the effect of PSA targeting by using an antibody with the same scaffold as previously used immunoconjugates but with random, non-specific, antigen binding region. This allows us to identify alterations generated by specific targeting and those related to passive bystander effects, such as enhanced permeability and retention (EPR). A control humanized IgG monoclonal antibody (hIgG1) and an isotype control IgG monoclonal antibody were conjugated with the chelator CHX-A"-DTPA. The immunoconjugate was radiolabeled with either Lutetium-177 ([177Lu]Lu) or Indium-111 ([111In]In). A biodistribution study in mice carrying LNCaP xenografts, was performed to evaluate the non-specific uptake of [177Lu]Lu-hIgG1 in tumors and normal organs. Further, therapy studies of [177Lu]Lu and [111In]In labeled IgG were performed in BALB/c mice carrying LNCaP xenografts. Tumor tissues of treated xenografts and control were sectioned and immunohistochemically stained for Ki67 and PSA. The highest tumor uptake for the [177Lu]Lu-hIgG1 was seen at 72 hours (7.2±2 %IA/g), when comparing the tumor uptake of the fPSA targeting antibody to the non-specific antibody, the non-specific antibody contributes to half of the tumor uptake at 72 h. The liver uptake was 3.1±0.5 %IA/g at 24 h, 2.8±0.5 %IA/g at 72 h and 1.3±0.6 %IA/g at 120 h in LNCaP xenografts, which was approximately three times lower at 24 h and two times lower at 72 h than for the antibody with preserved targeting. Immunohistochemical labeling showed a reduction of PSA expression and a reduction of Ki67 labeled cells in the [111In]In treated LNCaP tumors, compared to vehicle and [177Lu]Lu treated mice. In conclusion, we found that specific targeting might negatively influence normal organ uptake when targeting secreted antigens. Furthermore, different energy deposition i.e. linear energy transfer of a radionuclide might have diverse effects on receptor expression and cell proliferation in tumors.

Evaluation of recombinant human IGF-1/IGFBP-3 on intraventricular hemorrhage prevention and survival in the preterm rabbit pup model.

Insulin-like growth factor-1 (IGF-1) is essential for normal brain development and regulates processes of vascular maturation. The pathogenesis of intraventricular hemorrhage (IVH) relates to the fragility of the immature capillaries in the germinal matrix, and its inability to resist fluctuations in cerebral blood flow. In this work, using different experimental setups, we aimed to (i) establish an optimal time-point for glycerol-induction of IVH in relation to time-point of recombinant human (rh) IGF-1/rhIGFBP-3 administration, and (ii) to evaluate the effects of a physiologic replacement dose of rhIGF-1/rhIGFBP-3 on prevention of IVH and survival in the preterm rabbit pup. The presence of IVH was evaluated using high-frequency ultrasound and post-mortem examinations. In the first part of the study, the highest incidence of IVH (> 60%), occurred when glycerol was administered at the earliest timepoint, e.g., 6 h after birth. At later time-points (18 and 24 h) the incidence decreased substantially. In the second part of the study, the incidence of IVH and mortality rate following rhIGF-1/rhIGFBP-3 administration was not statistically different compared to vehicle treated animals. To evaluate the importance of maintaining intrauterine serum levels of IGF-1 following preterm birth, as reported in human interventional studies, additional studies are needed to further characterize and establish the potential of rhIGF-1/rhIGFBP-3 in reducing the prevalence of IVH and improving survival in the preterm rabbit pup.

Insulin-like growth factor 1 supplementation supports motor coordination and affects myelination in preterm pigs.

Introduction: Preterm infants have increased risk of impaired neurodevelopment to which reduced systemic levels of insulin-like growth factor 1 (IGF-1) in the weeks after birth may play a role. Hence, we hypothesized that postnatal IGF-1 supplementation would improve brain development in preterm pigs, used as a model for preterm infants. Methods: Preterm pigs delivered by cesarean section received recombinant human IGF-1/IGF binding protein-3 complex (rhIGF-1/rhIGFBP-3, 2.25 mg/kg/day) or vehicle from birth to postnatal day 19. Motor function and cognition were assessed by monitoring of in-cage and open field activities, balance beam test, gait parameters, novel object recognition and operant conditioning tests. Collected brains were subject to magnetic resonance imaging (MRI), immunohistochemistry, gene expression analyses and protein synthesis measurements. Results: The IGF-1 treatment increased cerebellar protein synthesis rates (both in vivo and ex vivo). Performance in the balance beam test was improved by IGF-1 but not in other neurofunctional tests. The treatment decreased total and relative caudate nucleus weights, without any effects to total brain weight or grey/white matter volumes. Supplementation with IGF-1 reduced myelination in caudate nucleus, cerebellum, and white matter regions and decreased hilar synapse formation, without effects to oligodendrocyte maturation or neuron differentiation. Gene expression analyses indicated enhanced maturation of the GABAergic system in the caudate nucleus (decreased NKCC1:KCC2 ratio) with limited effects in cerebellum or hippocampus. Conclusion: Supplemental IGF-1 during the first three weeks after preterm birth may support motor function by enhancing GABAergic maturation in the caudate nucleus, despite reduced myelination. Supplemental IGF-1 may support postnatal brain development in preterm infants, but more studies are required to identify optimal treatment regimens for subgroups of very or extremely preterm infants.

Insulin-Like Growth Factor-1 Supplementation Promotes Brain Maturation in Preterm Pigs.

Very preterm infants show low levels of insulin-like growth factor-1 (IGF-1), which is associated with postnatal growth restriction and poor neurologic outcomes. It remains unknown whether supplemental IGF-1 may stimulate neurodevelopment in preterm neonates. Using cesarean-delivered preterm pigs as a model of preterm infants, we investigated the effects of supplemental IGF-1 on motor function and on regional and cellular brain development. Pigs were treated with 2.25 mg/kg/d recombinant human IGF-1/IGF binding protein-3 complex from birth until day 5 or 9 before the collection of brain samples for quantitative immunohistochemistry (IHC), RNA sequencing, and quantitative PCR analyses. Brain protein synthesis was measured using in vivo labeling with [2H5] phenylalanine. We showed that the IGF-1 receptor was widely distributed in the brain and largely coexisted with immature neurons. Region-specific quantification of IHC labeling showed that IGF-1 treatment promoted neuronal differentiation, increased subcortical myelination, and attenuated synaptogenesis in a region-dependent and time-dependent manner. The expression levels of genes involved in neuronal and oligodendrocyte maturation, and angiogenic and transport functions were altered, reflecting enhanced brain maturation in response to IGF-1 treatment. Cerebellar protein synthesis was increased by 19% at day 5 and 14% at day 9 after IGF-1 treatment. Treatment had no effect on Iba1+ microglia or regional brain weights and did not affect motor development or the expression of genes related to IGF-1 signaling. In conclusion, the data show that supplemental IGF-1 promotes brain maturation in newborn preterm pigs. The results provide further support for IGF-1 supplementation therapy in the early postnatal period in preterm infants.

Characterization of choroid plexus in the preterm rabbit pup following subcutaneous administration of recombinant human IGF-1/IGFBP-3.

Insulin-like growth factor-1 (IGF-1) is essential for normal brain development and regulates essential processes of vascular maturation and stabilization. Importantly, preterm birth is associated with reduced serum levels of IGF-1 as compared to in utero levels. Using a preterm rabbit pup model, we investigated the uptake of systemic recombinant human (rh) IGF-1 in complex with its main binding protein IGF-binding protein 3 (BP-3) to the brain parenchyma via the choroid plexus. Five hours after subcutaneous administration, labeled rhIGF-1/rhIGFBP-3 displayed a widespread presence in the choroid plexus of the lateral and third ventricle, however, to a less degree in the fourth, as well as in the perivascular and subarachnoid space. We found a time-dependent uptake of IGF-1 in cerebrospinal fluid, decreasing with postnatal age, and a translocation of IGF-1 through the choroid plexus. The impact of systemic rhIGF-1/rhIGFBP-3 on IGF-1 receptor activation in the choroid plexus decreased with postnatal age, correlating with IGF-1 uptake in cerebrospinal fluid. In addition, choroid plexus gene expression was observed to increase with postnatal age. Moreover, using choroid plexus in vitro cell cultures, gene expression and protein synthesis were further investigated upon rhIGF-1/rhIGFBP-3 stimulation as compared to rhIGF-1 alone, and found not to be differently altered. Here, we characterize the uptake of systemic rhIGF-1/rhIGFBP-3 to the preterm brain, and show that the interaction between systemic rhIGF-1/rhIGFBP-3 and choroid plexus varies over time.

Distribution of aldehyde dehydrogenase 1-positive stem cells in benign mammary tissue from women with and without breast cancer.

AIMS: Aldehyde dehydrogenase 1 (ALDH1) in female breast tissue has been linked to stem cells, but little is known about the benign cellular organization in situ. We investigated the distribution of ALDH1-immunoreactive (ALDH1+) cells in histomorphologically benign breast tissue from 28 women with or without breast cancer. METHODS AND RESULTS: ALDH1+ cells were detected in benign tissue of women aged 20-72 years, located most commonly at the luminal and intermediate ductular levels and in the stroma. ALDH1+ cell populations and Ki67+ cell populations were present in separate ductules, both cell types rarely showing epithelial differentiation. ALDH1+ cells were non-reactive to Ki67 and oestrogen receptor. Stromal round/oval ALDH1+ non-leukocyte cells in both age groups expressed contractile protein. There was a lower concentration of luminal and intermediate ductular ALDH1+ cells in postmenopausal women than in premenopausal women, and in cancer patients than in non-cancer patients, and a higher concentration in women receiving exogenous hormones. CONCLUSIONS: This study provides further evidence for the stem cell character of ALDH1+ cells, here in benign breast tissue of cancer and non-cancer patients throughout non-lactating adult life, and contributes evidence of benign stromal ALDH1+ cells. The distribution of ductular ALDH1+ stem cells appears to be influenced by hormonal status.

Reversible Self-Assembled Monolayers with Tunable Surface Dynamics for Controlling Cell Adhesion Behavior.

Cells adhering onto surfaces sense and respond to chemical and physical surface features. The control over cell adhesion behavior influences cell migration, proliferation, and differentiation, which are important considerations in biomaterial design for cell culture, tissue engineering, and regenerative medicine. Here, we report on a supramolecular-based approach to prepare reversible self-assembled monolayers (rSAMs) with tunable lateral mobility and dynamic control over surface composition to regulate cell adhesion behavior. These layers were prepared by incubating oxoacid-terminated thiol SAMs on gold in a pH 8 HEPES buffer solution containing different mole fractions of ω-(ethylene glycol)2-4- and ω-(GRGDS)-, α-benzamidino bolaamphiphiles. Cell shape and morphology were influenced by the strength of the interactions between the amidine-functionalized amphiphiles and the oxoacid of the underlying SAMs. Dynamic control over surface composition, achieved by the addition of inert filler amphiphiles to the RGD-functionalized rSAMs, reversed the cell adhesion process. In summary, rSAMs featuring mobile bioactive ligands offer unique capabilities to influence and control cell adhesion behavior, suggesting a broad use in biomaterial design, tissue engineering, and regenerative medicine.

Acute pyelonephritis and renal scarring are caused by dysfunctional innate immunity in mCxcr2 heterozygous mice.

The CXCR1 receptor and chemokine CXCL8 (IL-8) support neutrophil-dependent clearance of uropathogenic Escherichia coli from the urinary tract. CXCR1 is reduced in children prone to pyelonephritis, and heterozygous hCXCR1 polymorphisms are more common in this patient group than in healthy individuals, strongly suggesting a disease association. Since murine CXCR2 (mCXCR2) is functionally similar to human CXCR1, we determined effects of gene heterozygosity on the susceptibility to urinary tract infection by infecting heterozygous (mCxcr2(+/-)) mice with uropathogenic Escherichia coli. Clearance of infection and tissue damage were assessed as a function of innate immunity in comparison to that in knockout (mCxcr2(-/-)) and wild-type (mCxcr2(+/+)) mice. Acute sepsis-associated mortality was increased and bacterial clearance drastically impaired in heterozygous compared to wild-type mice. Chemokine and neutrophil responses were delayed along with evidence of neutrophil retention and unresolved kidney inflammation 1 month after infection. This was accompanied by epithelial proliferation and subepithelial fibrosis. The heterozygous phenotype was intermediate, between knockout and wild-type mice, but specific immune cell infiltrates that accompany chronic infection in knockout mice were not found. Hence, the known heterozygous CXCR1 polymorphisms may predispose patients to acute pyelonephritis and urosepsis.

Biodistribution of fluorescence-labelled EGF protein from slow release NanoZolid depots in mouse.

AIM: The study was designed to evaluate the ability of the calcium sulfate based NanoZolid® drug delivery technology to locally release the epidermal growth factor (EGF) protein while maintaining its biological activity. METHODS: NanoZolid-formulated EGF protein labelled with a near infrared dye (EGF-NIR) depots or EGF-NIR dissolved in PBS were injected subcutaneously into mice bearing EGF receptor (EGFR) positive human A549 lung cancer tumors inoculated subcutaneously. The release and biodistribution of the EGF-NIR were investigated in vivo longitudinally up to 96 h post administration, utilizing whole body fluorescence imaging. In order to confirm the in vivo findings, histological analysis of tumor cryosections was performed to investigate EGF-NIR fluorescent signal and EGFR expression level by immunofluorescence labelling. RESULTS: The in vivo fluorescence imaging showed a controlled release profile of the EGF-NIR loaded in the NanoZolid depots compared to free EGF-NIR. Histological analysis of the tumors further demonstrated a prevailing distribution of EGF-NIR in regions with high levels of EGFR expression. CONCLUSION: Calcium sulfate based depots can be used to formulate EGF while maintaining its biological activity, e.g. receptor binding capability. This may have a good clinical potential for local delivery of biomolecules to enhance treatment efficacy and minimize systemic adverse effects.

A Conjugation Strategy to Modulate Antigen Binding and FcRn Interaction Leads to Improved Tumor Targeting and Radioimmunotherapy Efficacy with an Antibody Targeting Prostate-Specific Antigen.

BACKGROUND: The humanized monoclonal antibody (mAb) hu5A10 specifically targets and internalizes prostate cancer cells by binding to prostate specific antigen (PSA). Preclinical evaluations have shown that hu5A10 is an excellent vehicle for prostate cancer (PCa) radiotheranostics. We studied the impact of different chelates and conjugation ratios on hu5A10's target affinity, neonatal fc-receptor interaction on in vivo targeting efficacy, and possible enhanced therapeutic efficacy. METHODS: In our experiment, humanized 5A10 (hu5A10) was conjugated with DOTA or DTPA at a molar ratio of 3:1, 6:1, and 12:1. Surface plasmon resonance (SPR) was used to study antigen and FcRn binding to the antibody conjugates. [111In]hu5A10 radio-immunoconjugates were administered intravenously into BALB/c mice carrying subcutaneous LNCaP xenografts. Serial Single-photon emission computed tomography (SPECT) images were obtained during the first week. Tumors were harvested and radionuclide distribution was analyzed by autoradiography along with microanatomy and immunohistochemistry. RESULTS: As seen by SPR, the binding to PSA was clearly affected by the chelate-to-antibody ratio. Similarly, FcRn (neonatal fc-receptor) interacted less with antibodies conjugated at high ratios of chelator, which was more pronounced for DOTA conjugates. The autoradiography data indicated a higher distribution of radioactivity to the rim of the tumor for lower ratios and a more homogenous distribution at higher ratios. Mice injected with ratio 3:1 111In-DOTA-hu5A10 showed no significant difference in tumor volume when compared to mice given vehicle over a time period of 3 weeks. Mice given a similar injection of ratio 6:1 111In-DOTA-hu5A10 or 6:1 111In-DTPA-hu5A10 or 12:1 111In-DTPA-hu5A10 showed significant tumor growth retardation. Conclusions: The present study demonstrated that the radiolabeling strategy could positively modify the hu5A10's capacity to bind PSA and complex with the FcRn-receptor, which resulted in more homogenous activity distribution in tumors and enhanced therapy efficacy.

HAMLET treatment delays bladder cancer development.

PURPOSE: HAMLET is a protein-lipid complex that kills different types of cancer cells. Recently we observed a rapid reduction in human bladder cancer size after intravesical HAMLET treatment. In this study we evaluated the therapeutic effect of HAMLET in the mouse MB49 bladder carcinoma model. MATERIALS AND METHODS: Bladder tumors were established by intravesical injection of MB49 cells into poly L-lysine treated bladders of C57BL/6 mice. Treatment groups received repeat intravesical HAMLET instillations and controls received alpha-lactalbumin or phosphate buffer. Effects of HAMLET on tumor size and putative apoptotic effects were analyzed in bladder tissue sections. Whole body imaging was used to study HAMLET distribution in tumor bearing mice compared to healthy bladder tissue. RESULTS: HAMLET caused a dose dependent decrease in MB49 cell viability in vitro. Five intravesical HAMLET instillations significantly decreased tumor size and delayed development in vivo compared to controls. TUNEL staining revealed selective apoptotic effects in tumor areas but not in adjacent healthy bladder tissue. On in vivo imaging Alexa-HAMLET was retained for more than 24 hours in the bladder of tumor bearing mice but not in tumor-free bladders or in tumor bearing mice that received Alexa-alpha-lactalbumin. CONCLUSIONS: Results show that HAMLET is active as a tumoricidal agent and suggest that topical HAMLET administration may delay bladder cancer development.

Distribution, cellular localization, and therapeutic potential of the tumor-associated antigen Ku70/80 in glioblastoma multiforme.

Antibodies specifically targeting tumor-associated antigens have proved to be important tools in the treatment of human cancer. A desirable target antigen should be unique to tumor cells, abundantly expressed, and readily available for antibody binding. The Ku70/80 DNA-repair protein is expressed in the nucleus of most cells; it is, however, also present on the cell surface of tumor cell lines, and antibodies binding Ku70/80 at the cell surface were recently shown to internalize into tumor cells. To evaluate the potential of Ku70/80-antigen as a therapeutic target for immunotoxins in glioblastoma multiforme, we investigated binding and localization of Ku70/80-specific antibodies in tissue samples from glioblastomas and normal human brains, and in glioma cell cultures. Furthermore, the internalization and drug-delivery capacity were evaluated by use of immunotoxicity studies. We demonstrate that Ku70/80 is localized on the cell plasma membrane of glioma cell lines, and is specifically present in human glioblastoma tissue. Antibodies bound to the Ku70/80 antigen on the cell surface of glioma cells were found to internalize via endocytosis, and shown to efficiently deliver toxins into glioblastoma cells. The data further imply that different antibodies directed against Ku70/80 possess different abilities to target the antigen, in relation to its presentation on the cell surface or intracellular localization. We conclude that Ku70/80 antigen is uniquely presented on the plasma membrane in glioblastomas, and that antibodies specific against the antigen have the capacity to selectively bind, internalize, and deliver toxins into tumor cells. These results imply that Ku70/80 is a potential target for immunotherapy of glioblastoma multiforme.