Beyond scattering and absorption: Perceptual unmixing of translucent liquids.

Is perception of translucence based on estimations of scattering and absorption of light or on statistical pseudocues associated with familiar materials? We compared perceptual performance with real and computer-generated stimuli. Real stimuli were glasses of milky tea. Milk predominantly scatters light and tea absorbs it, but since the tea absorbs less as the milk concentration increases, the effects of milkiness and strength on scattering and absorption are not independent. Conversely, computer-generated stimuli were glasses of "milky tea" in which absorption and scattering were independently manipulated. Observers judged tea concentrations regardless of milk concentrations, or vice versa. Maximum-likelihood conjoint measurement was used to estimate the contributions of each physical component-concentrations of milk and tea, or amounts of scattering and absorption-to perceived milkiness or tea strength. Separability of the two physical dimensions was better for real than for computer-generated teas, suggesting that interactions between scattering and absorption were correctly accounted for in perceptual unmixing, but unmixing was always imperfect. Since the real and rendered stimuli represent different physical processes and therefore differ in their image statistics, perceptual judgments with these stimuli allowed us to identify particular pseudocues (presumably learned with real stimuli) that explain judgments with both stimulus sets.

Pharmacokinetic and Metabolomic Studies with a Promising Radiation Countermeasure, BBT-059 (PEGylated interleukin-11), in Rhesus Nonhuman Primates.

BBT-059, a long-acting PEGylated interleukin-11 (IL-11) analog that is believed to have hematopoietic promoting and anti-apoptotic properties, is being developed as a potential radiation medical countermeasure (MCM) for hematopoietic acute radiation syndrome (H-ARS). This agent has been shown to improve survival in lethally irradiated mice. To further evaluate the drug's toxicity and safety profile, 12 naïve nonhuman primates (NHPs, rhesus macaques) were administered one of three doses of BBT-059 subcutaneously and were monitored for the next 21 days. Blood samples were collected throughout the study to assess the pharmacokinetics (PK) and pharmacodynamics (PD) of the drug as well as its effects on complete blood counts, cytokines, vital signs, and to conduct metabolomic studies. No adverse effects were detected in any treatment group during the study. Short-term changes in metabolomic profiles were present in all groups treated with BBT-059 beginning immediately after drug administration and reverting to near normal levels by the end of the study period. Several pathways and metabolites, particularly those related to inflammation and steroid hormone biosynthesis, were activated by BBT-059 administration. Taken together, these observations suggest that BBT-059 has a good safety profile for further development as a radiation MCM for regulatory approval for human use.

Interleukin-11 protects against renal ischemia and reperfusion injury.

Renal ischemia reperfusion (IR) injury causes renal tubular necrosis, apoptosis, and inflammation leading to acute and chronic kidney dysfunction. IL-11 is a multifunctional hematopoietic cytokine clinically approved to treat chemotherapy-induced thrombocytopenia. Recent studies suggest that IL-11 also has potent antiapoptotic and antinecrotic properties. In this study, we tested the hypothesis that exogenous IL-11 protects against renal IR injury and determined the mechanisms involved in renal protection. Pretreatment with human recombinant IL-11 (HR IL-11) or with long-acting site-specific polyethylene glycol (PEG)-conjugated human IL-11 analog (PEGylated IL-11) produced partial but significant protection against renal IR injury in mice. In addition, HR IL-11 or PEGylated IL-11 given 30-60 min after IR also provided renal protection in mice. Significant reductions in renal tubular necrosis and neutrophil infiltration as well as tubular apoptosis were observed in mice treated with HR IL-11 or PEGylated IL-11. Furthermore, HR IL-11 or PEGylated IL-11 decreased both necrosis and apoptosis in human proximal tubule (HK-2) cells in culture. Mechanistically, IL-11 increased nuclear translocation of hypoxia-inducible factor-1α (HIF-1α) and induced sphingosine kinase-1 (SK1) expression and activity in HK-2 cells. Moreover, selective HIF-1α inhibitors blocked IL-11-mediated induction of SK1 in HK-2 cells. Finally, HR IL-11 or PEGylated IL-11 failed to protect against renal IR injury in SK1-deficient mice. Together, our data show powerful renal protective effects of exogenous IL-11 against IR injury by reducing necrosis, inflammation, and apoptosis through induction of SK1 via HIF-1α.

Age and Sex Divergence in Hematopoietic Radiosensitivity in Aged Mouse Models of the Hematopoietic Acute Radiation Syndrome.

The hematopoietic system is highly sensitive to stress from both aging and radiation exposure, and the hematopoietic acute radiation syndrome (H-ARS) should be modeled in the geriatric context separately from young for development of age-appropriate medical countermeasures (MCMs). Here we developed aging murine H-ARS models, defining radiation dose response relationships (DRRs) in 12-month-old middle-aged and 24-month-old geriatric male and female C57BL/6J mice, and characterized diverse factors affecting geriatric MCM testing. Groups of approximately 20 mice were exposed to ∼10 different doses of radiation to establish radiation DRRs for estimation of the LD50/30. Radioresistance increased with age and diverged dramatically between sexes. The LD50/30 in young adult mice averaged 853 cGy and was similar between sexes, but increased in middle age to 1,005 cGy in males and 920 cGy in females, with further sex divergence in geriatric mice to 1,008 cGy in males but 842 cGy in females. Correspondingly, neutrophils, platelets, and functional hematopoietic progenitor cells were all increased with age and rebounded faster after irradiation. These effects were higher in aged males, and neutrophil dysfunction was observed in aged females. Upstream of blood production, hematopoietic stem cell (HSC) markers associated with age and myeloid bias (CD61 and CD150) were higher in geriatric males vs. females, and sex-divergent gene signatures were found in HSCs relating to cholesterol metabolism, interferon signaling, and GIMAP family members. Fluid intake per gram body weight decreased with age in males, and decreased after irradiation in all mice. Geriatric mice of substrain C57BL/6JN sourced from the National Institute on Aging were significantly more radiosensitive than C57BL/6J mice from Jackson Labs aged at our institution, indicating mouse source and substrain should be considered in geriatric radiation studies. This work highlights the importance of sex, vendor, and other considerations in studies relating to hematopoiesis and aging, identifies novel sex-specific functional and molecular changes in aging hematopoietic cells at steady state and after irradiation, and presents well-characterized aging mouse models poised for MCM efficacy testing for treatment of acute radiation effects in the elderly.

Reamer-irrigator-aspirator indications and clinical results: a systematic review.

BACKGROUND: The 'reamer-irrigator-aspirator' (RIA) is an innovation developed to reduce fat embolism (FE) and thermal necrosis (TN) that can occur during reaming/nailing of long-bone fractures. Since its inception its indications have expanded to include the treatment of long-bone osteomyelitis and as a harvester of bone graft/mesenchymal stem cells (MSCs). METHODS: This study involved a systematic review, via Pubmed® and Google Scholar®, of English language sources (nine non-clinical studies, seven clinical studies and seven case reports) using the keywords: 'reamer', 'irrigator', 'aspirator' (1st May 2010). Sources were reviewed with reference to the RIAs efficacy in (1) preventing FE/TN, (2) treating long-bone osteomyelitis, (3) harvesting bone graft/MSCs, and (4) operating safely. Experimental data supports the use of the RIA in preventing FE and TN, however, there is a paucity of clinical data. CONCLUSIONS: The RIA is a reliable method in achieving high volumes of bone graft/MSCs, and high union rates are reported when using RIA bone-fragments to treat non-unions. Evidence suggests possible effectiveness in treating long-bone osteomyelitis. The RIA appears relatively safe, with a low rate of morbidity provided a meticulous technique is used. When complications occur they respond well to conventional techniques. The RIA demands further investigation especially with respect to the optimal application of MSCs for bone repair strategies.

Focal Myositis of the Deltoid Muscle: The Role of Magnetic Resonance Imaging for Diagnosis.

Introduction: Focal myositis is a rare condition first described by Heffner et al., in 1977, as a self-limiting condition of unknown aetiology. It presents as an inflammatory pseudo tumour in skeletal muscle and can present diagnostic difficulty, being commonly mistaken for tissue of vascular, inflammatory, or neoplastic origin. Diagnosis is traditionally confirmed by muscle biopsy. We present a case where magnetic resonance imaging (MRI) was used to confirm the diagnosis without need for biopsy. Case Presentation: A 19-year year-old female presented with a two2-year history of intermittent swelling of the deltoid associated with pain and tenderness to palpation. . There was no history of trauma or systemic illness. . She was symptomatic with pain, swelling, and tenderness over the left deltoid with no restriction in range of movement of the shoulder or neck. Plain radiographs were normal and MRI magnetic resonance imaging showed diffuse odeamatousedematous signal changes on the proton density weighted sequence within the deltoid muscle and no plexiform neurofibroma. Nerve conduction and electromyography studies were within normal limits excluding an axillary nerve lesion. The patient underwent extensive screening for connective tissue disorders and creatine kinase and lactate dehydrogenase levels were within limits. The patient underwent neuromuscular specialist review confirming that this appeared to be a rare case of focal myositis in the deltoid. . The serial MRI scans confirmed resolution of the condition. Conclusion: Focal myositis of the deltoid is a rare cause of shoulder pain. . We have shown that sequential MRI scanning can obviate the need for muscle biopsy, which has historically been required for diagnostic confirmation. The MRI appearance on the proton density weighted sequence showed diffuse odeamatousedematous signal changes and no plexiform neurofibroma within the deltoid and is a description that has not been previously used for this rare diagnosis.

Polypharmacy to Mitigate Acute and Delayed Radiation Syndromes.

There is a need for countermeasures to mitigate lethal acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE). In WAG/RijCmcr rats, ARS occurs by 30-days following total body irradiation (TBI), and manifests as potentially lethal gastrointestinal (GI) and hematopoietic (H-ARS) toxicities after >12.5 and >7 Gy, respectively. DEARE, which includes potentially lethal lung and kidney injuries, is observed after partial body irradiation >12.5 Gy, with one hind limb shielded (leg-out PBI). The goal of this study is to enhance survival from ARS and DEARE by polypharmacy, since no monotherapy has demonstrated efficacy to mitigate both sets of injuries. For mitigation of ARS following 7.5 Gy TBI, a combination of three hematopoietic growth factors (polyethylene glycol (PEG) human granulocyte colony-stimulating factor (hG-CSF), PEG murine granulocyte-macrophage-CSF (mGM-CSF), and PEG human Interleukin (hIL)-11), which have shown survival efficacy in murine models of H-ARS were tested. This triple combination (TC) enhanced survival by 30-days from ∼25% to >60%. The TC was then combined with proven medical countermeasures for GI-ARS and DEARE, namely enrofloxacin, saline and the angiotensin converting enzyme inhibitor, lisinopril. This combination of ARS and DEARE mitigators improved survival from GI-ARS, H-ARS, and DEARE after 7.5 Gy TBI or 13 Gy PBI. Circulating blood cell recovery as well as lung and kidney function were also improved by TC + lisinopril. Taken together these results demonstrate an efficacious polypharmacy to mitigate radiation-induced ARS and DEARE in rats.

Corrigendum: Polypharmacy to Mitigate Acute and Delayed Radiation Syndromes.

[This corrects the article DOI: 10.3389/fphar.2021.634477.].

Characterization of a Long-Acting Site-Specific PEGylated Murine GM-CSF Analog and Analysis of Its Hematopoietic Properties in Normal and Cyclophosphamide-Treated Neutropenic Rats.

Previously we reported that site-specific modification of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) A3C analog with polyethylene glycol (PEG) dramatically improved the pharmacokinetic properties of the protein in rats. However, we could not evaluate the hematological properties of the PEG-A3C protein in rats because human GM-CSF is inactive in rodents. To study the biological effects of PEGylated GM-CSF analogs in rodents we created a homologous site-specific PEGylated murine (mu) GM-CSF (T3C) protein. muGM-CSF and the T3C protein were expressed in Escherichia coli and purified by column chromatography. The purified T3C protein was covalently modified with a linear 20 kDa- or a branched 40 kDa-maleimide-PEG, and the monoPEGylated proteins purified by column chromatography. muGM-CSF, T3C and the two PEG-T3C proteins had comparable in vitro biological activities, as measured by stimulation of proliferation of the murine FDC-P1 cell line. The PEG-T3C proteins had 10- to 25-fold longer circulating half-lives than muGM-CSF and stimulated greater and longer lasting increases in neutrophils and white blood cells than muGM-CSF following a single intravenous or subcutaneous administration to rats. Treatment of rats made neutropenic with cyclophosphamide with the PEG-T3C proteins shortened the time for recovery of neutrophils to normal levels from 9 or 10 days to 5 or 6 days, whereas muGM-CSF showed no benefit versus vehicle solution. Acceleration of neutrophil recovery in cyclophosphamide-treated rats required a minimum of three PEG-T3C treatments over five days. The PEG-T3C proteins should prove useful for evaluating the potential therapeutic benefits of GM-CSF and long-acting GM-CSF proteins in rodent disease models.

Transient Existence of Circulating Mesenchymal Stem Cells in the Deep Veins in Humans Following Long Bone Intramedullary Reaming.

The biology of mesenchymal stem cells (MSCs) in humans is incompletely understood and a possible role of systemically circulating cells in health and autoimmune disease remains controversial. Physiological movement of bone marrow MSCs to sites of injury would support the rationale for intravenous administration for relocation to damaged organs. We hypothesized that biophysical skeletal trauma rather than molecular cues may explain reported MSC circulation phenomena. Deep-femoral vein (FV) and matched peripheral vein blood samples (PVBs) were collected from patients undergoing lower-limb orthopaedic procedures during surgery (tibia using conventional sequential reaming, n = 9, femur using reamer/irrigator/aspirator (RIA), n = 15). PVBs were also taken from early (n = 15) and established (n = 12) rheumatoid arthritis (RA) patients and healthy donors (n = 12). Colony-forming unit-fibroblasts (CFU-Fs) were found in 17/36 FVBs but only 7/74 PVBs (mostly from femoral RIA); highly proliferative clonogenic cells were not generated. Only one colony was found in control/RA samples (n = 28). The rare CFU-Fs' MSC nature was confirmed by phenotypic: CD105+/CD73+/CD90+ and CD19-/CD31-/CD33-/CD34-/CD45-/CD61-, and molecular profiles with 39/80 genes (including osteo-, chondro-, adipo-genic and immaturity markers) similar across multiple MSC tissue controls, but not dermal fibroblasts. Analysis of FVB-MSCs suggested that their likely origin was bone marrow as only two differences were observed between FVB-MSCs and IC-BM-MSCs (ACVR2A, p = 0.032 and MSX1, p = 0.003). Stromal cells with the phenotype and molecular profile of MSCs were scarcely found in the circulation, supporting the hypothesis that their very rare presence is likely linked to biophysical micro-damage caused by skeletal trauma (here orthopaedic manipulation) rather than specific molecular cues to a circulatory pool of MSCs capable of repair of remote organs or tissues. These findings support the use of organ resident cells or MSCs placed in situ to repair tissues rather than systemic administration.

Delayed effects of acute whole body lethal radiation exposure in mice pre-treated with BBT-059.

The threat of nuclear exposure is heightened and it is imperative to identify potential countermeasures for acute radiation syndrome. Currently no countermeasures have been approved for prophylactic administration. Effective countermeasures should function to increase survival in the short term as well as to increase the overall prognosis of an exposed individual long term. Here we describe the use of a promising radiation countermeasure, BBT-059, and the results of a long term mouse study (up to 12 months) in the male CD2F1 strain using 60Co gamma irradiation (~0.6 Gy/min, 7.5-12.5 Gy). We report the dose reduction factor of 1.28 for BBT-059 (0.3 mg/kg) compared to control administered 24 h prior to irradiation. In the long term study animals showed accelerated recovery in peripheral blood cell counts, bone marrow colony forming units, sternal cellularity and megakaryocyte numbers in drug treated mice compared to formulation buffer. In addition, increased senescence was observed in the kidneys of animals administered control or drug and exposed to the highest doses of radiation. Decreased levels of E-cadherin, LaminB1 and increased levels of Cyc-D and p21 in spleen lysates were observed in animals administered control. Taken together the results indicate a high level of protection following BBT-059 administration in mice exposed to lethal and supralethal doses of total body gamma-radiation.

IFN-γ-tethered hydrogels enhance mesenchymal stem cell-based immunomodulation and promote tissue repair.

Because of their immunomodulatory activities, human mesenchymal stem cells (hMSCs) are being explored to treat a variety of chronic conditions such as inflammatory bowel disorders and graft-vs-host disease. Treating hMSCs with IFN-γ prior to administration augments these immunomodulatory properties; however, this ex vivo treatment limits the broad applicability of this therapy due to technical and regulatory issues. In this study, we engineered an injectable synthetic hydrogel with tethered recombinant IFN-γ that activates encapsulated hMSCs to increase their immunomodulatory functions and avoids the need for ex vivo manipulation. Tethering IFN-γ to the hydrogel increases retention of IFN-γ within the biomaterial while preserving its biological activity. hMSCs encapsulated within hydrogels with tethered IFN-γ exhibited significant differences in cytokine secretion and showed a potent ability to halt activated T-cell proliferation and monocyte-derived dendritic cell differentiation compared to hMSCs that were pre-treated with IFN-γ and untreated hMSCs. Importantly, hMSCs encapsulated within hydrogels with tethered IFN-γ accelerated healing of colonic mucosal wounds in both immunocompromised and immunocompetent mice. This novel approach for licensing hMSCs with IFN-γ may enhance the clinical translation and efficacy of hMSC-based therapies.

Mitochondrial Degeneration and Autophagy Associated With Delayed Effects of Radiation in the Mouse Brain.

Mitochondria are linked with various radiation responses, including mitophagy, genomic instability, apoptosis, and the bystander effect. Mitochondria play an important role in preserving cellular homeostasis during stress responses, and dysfunction in mitochondrial contributes to aging, carcinogenesis and neurologic diseases. In this study, we have investigated the mitochondrial degeneration and autophagy in the hippocampal region of brains from mice administered with BBT-059, a long-acting interleukin-11 analog, or its formulation buffer 24 h prior to irradiation at different radiation doses collected at 6 and 12 months post-irradiation. The results demonstrated a higher number of degenerating mitochondria in 12 Gy BBT-059 treated mice after 6 months and 11.5 Gy BBT-059 treated mice after 12 months as compared to the age-matched naïve (non-irradiated control animals). Apg5l, Lc3b and Sqstm1 markers were used to analyze the autophagy in the brain, however only the Sqstm1 marker exhibited significantly reduced expression after 12 months in 11.5 Gy BBT-059 treated mice as compared to naïve. Immunohistochemistry (IHC) results of Bcl2 also demonstrated a decrease in expression after 12 months in 11.5 Gy BBT-059 treated mice as compared to other groups. In conclusion, our results demonstrated that higher doses of ionizing radiation (IR) can cause persistent upregulation of mitochondrial degeneration. Reduced levels of Sqstm1 and Bcl2 can lead to intensive autophagy which can lead to degradation of cellular structure.

Compartment syndrome with an isolated Salter Harris II fracture of the distal tibia.

A 14-year-old boy sustained a Salter Harris II fracture to his right distal tibia after a fall from his skateboard. He rapidly went on to develop the signs and symptoms of compartment syndrome, and he underwent emergency fasciotomy. This resulted in relief of his symptoms. After this procedure, his fracture was fixed with a single anteroposterior screw. He made a full and uncomplicated recovery, with no clinical or radiological evidence of epiphyseal growth arrest.

PEGylated IL-11 (BBT-059): A Novel Radiation Countermeasure for Hematopoietic Acute Radiation Syndrome.

Interleukin-11 was developed to reduce chemotherapy-induced thrombocytopenia; however, its clinical use was limited by severe adverse effects in humans. PEGylated interleukin-11 (BBT-059), developed by Bolder Biotechnology, Inc., exhibited a longer half-life in rodents and induced longer-lasting increases in hematopoietic cells than interleukin-11. A single dose of 1.2 mg kg of BBT-059, administered subcutaneously to CD2F1 mice (12-14 wk, male) was found to be safe in a 14 d toxicity study. The drug demonstrated its efficacy both as a prophylactic countermeasure and a mitigator in CD2F1 mice exposed to Co gamma total-body irradiation. A single dose of 0.3 mg kg, administered either 24 h pre-, 4 h post-, or 24 h postirradiation increased the survival of mice to 70-100% from lethal doses of radiation. Preadministration (-24 h) of the drug conferred a significantly (p < 0.05) higher survival compared to 24 h post-total-body irradiation. There was significantly accelerated recovery from radiation-induced peripheral blood neutropenia and thrombocytopenia in animals pretreated with BBT-059. The drug also increased bone marrow cellularity and megakaryocytes and accelerated multilineage hematopoietic recovery. In addition, BBT-059 inhibited the induction of radiation-induced hematopoietic biomarkers, thrombopoietin, erythropoietin, and Flt-3 ligand. These results indicate that BBT-059 is a promising radiation countermeasure, demonstrating its potential to be used both pre- and postirradiation for hematopoietic acute radiation syndrome with a broad window for medical management in a radiological or nuclear event.

A long-acting, mono-PEGylated human growth hormone analog is a potent stimulator of weight gain and bone growth in hypophysectomized rats.

Recombinant human GH is used to treat GH deficiency in children and adults and wasting in AIDS patients. GH has a circulating half-life of only a few hours in humans and must be administered to patients by daily injection for maximum effectiveness. Previous studies showed that longer-acting forms of GH could be created by modification of GH with multiple 5-kDa amine-reactive polyethylene glycols (PEGs). Eight of nine lysine residues and the N-terminal amino acid were modified to varying extents by amine PEGylation of GH. The amine-PEGylated GH product comprised a complex mixture of multiple PEGylated species that differed from one another in mass, in vitro bioactivity, and in vivo potency. In vitro bioactivity of GH was reduced 100- to 1000-fold by extensive amine PEGylation of the protein. Here we describe a homogeneously modified, mono-PEGylated GH protein that possesses near complete in vitro bioactivity, a long half-life, and increased potency in vivo. The mono-PEGylated GH was created by substituting cysteine for threonine-3 (T3C) of GH, followed by modification of the added cysteine residue with a single 20-kDa cysteine-reactive PEG. The PEG-T3C protein has an approximate 8-fold longer half-life than GH after sc administration to rats. Every other day or every third day administration of PEG-T3C stimulates increases in body weight and tibial epiphysis growth comparable with that produced by daily administration of GH in hypophysectomized rats. Long-acting, mono-PEGylated GH analogs such as PEG-T3C are promising candidates for future testing in humans.

Design of homogeneous, monopegylated erythropoietin analogs with preserved in vitro bioactivity.

OBJECTIVE: Erythropoietin (Epo) bioactivity is significantly reduced by modification of lysine residues with amine-reactive reagents, which are the most commonly used reagents for attaching polyethylene glycols (PEGs) to proteins to improve protein half-life in vivo. The aims of this study were to determine whether Epo bioactivity can be preserved by targeting attachment of maleimide-PEGs to engineered cysteine analogs of Epo, and to determine whether the pegylated Epo cysteine analogs have improved pharmacokinetic properties in vivo. MATERIALS AND METHODS: Thirty-four Epo cysteine analogs were constructed by site-directed mutagenesis and expressed as secreted proteins in baculovirus-infected insect cells. Following purification, monopegylated derivatives of 12 cysteine analogs were prepared using 20-kDa maleimide-PEGs. In vitro biological activities of the proteins were measured in an Epo-dependent cell proliferation assay. Plasma levels of insect cell-expressed wild-type Epo (BV Epo) and a pegylated Epo cysteine analog were quantitated by ELISA following intravenous administration to rats. RESULTS: Biological activities of 17 purified Epo cysteine analogs and 10 purified pegylated Epo cysteine analogs were comparable to that of BV Epo in the in vitro bioassay. The only pegylated cysteine analogs that displayed consistently reduced in vitro bioactivities were substitutions for lysine residues, PEG-K45C and PEG-K154C. The pegylated Epo cysteine analog had a slower initial distribution phase and a longer terminal half-life than BV Epo in rats, but the majority of both proteins were cleared rapidly from the circulation. CONCLUSIONS: Targeted attachment of maleimide-PEGs to engineered Epo cysteine analogs permits rational design of monopegylated Epo analogs with minimal loss of in vitro biological activity. Insect cell-expressed Epo proteins are cleared rapidly from the circulation in rats, possibly due to improper glycosylation. Site-specific pegylation appears to improve the pharmacokinetic properties of Epo.

Enhanced circulating half-life and hematopoietic properties of a human granulocyte colony-stimulating factor/immunoglobulin fusion protein.

OBJECTIVE: The aim of this study was to determine whether fusion proteins comprising human granulocyte colony-stimulating factor (G-CSF) joined to human immunoglobulin G1 and G4 (IgG1 and IgG4) Fc and C(H) domains are biologically active and have improved pharmacokinetic and hematopoietic properties in vivo. MATERIAL AND METHODS: Chimeric genes encoding human G-CSF fused to the N-termini of the Fc and C(H) domains of human IgG1 and IgG4 were constructed and used to transfect monkey COS cells. The fusion proteins were purified from the conditioned media by protein A affinity chromatography. Bioactivities of the proteins were measured in a G-CSF-dependent in vitro bioassay. Pharmacokinetic and granulopoietic properties of the G-CSF/IgG1-Fc fusion protein were measured in normal rats. RESULTS: The G-CSF/IgG-Fc and G-CSF/IgG-C(H) fusion proteins were secreted from transfected COS cells primarily as disulfide-linked homodimers. On a molar basis, the purified G-CSF/IgG-Fc fusion proteins were as active as G-CSF in in vitro bioassays, whereas bioactivities of the purified G-CSF/IgG-C(H) fusion proteins were decreased 3- to 4-fold. The G-CSF/IgG1-Fc fusion protein displayed a slower plasma clearance rate and stimulated greater and longer lasting increases in circulating neutrophils and white blood cells than G-CSF following intravenous and subcutaneous administration to rats. CONCLUSION: Fusion of G-CSF to human IgG domains results in homodimeric fusion proteins possessing high in vitro bioactivities, long circulating half-lives, and enhanced hematopoietic properties in vivo.

The changing face(book) of psychiatry: can we justify 'following' patients' social media activity?

Individuals with mental health issues may post information on social networking sites that can provide an insight into their mental health status. It could be argued that doctors (and specifically psychiatrists) should understand the way in which social media is used by their patients to gain a better insight into their illnesses. However, choosing to actively monitor a patient's social media activity raises important questions about the way in which medical students, qualified clinicians and other healthcare professionals obtain information about patients. While this may be framed as a mere form of 'collateral history-taking', there are obvious practical and ethical problems with doing so. Here, a case is made against monitoring the social media activity of patients involved with psychiatric services.

Cytokine refacing effect reduces granulocyte macrophage colony-stimulating factor susceptibility to antibody neutralization.

Crohn's Disease (CD) afflicts over half a million Americans with an annual economic impact exceeding $10 billion. Granulocyte macrophage colony-stimulating factor (GM-CSF) can increase patient immune responses against intestinal microbes that promote CD and has been effective for some patients in clinical trials. We have made important progress toward developing GM-CSF variants that could be more effective CD therapeutics by virtue of being less prone to neutralization by the endogenous GM-CSF autoantibodies that are highly expressed in CD patients. Yeast display engineering revealed mutations that increase GM-CSF variant binding affinity by up to ∼3-fold toward both GM-CSF receptor alpha and beta subunits in surface plasmon resonance experiments. Increased binding affinity did not reduce GM-CSF half-maximum effective concentration (EC50) values in conventional in vitro human leukocyte proliferation assays. Affinity-enhancing mutations did, however, promote a 'refacing effect' that imparted all five evaluated GM-CSF variants with increased in vitro bioactivity in the presence of GM-CSF-neutralizing polyclonal antisera. The most improved variant, H15L/R23L, was 6-fold more active than wild-type GM-CSF. Incorporation of additional known affinity-increasing mutations could augment the refacing effect and concomitant bioactivity improvements described here.