Double versus Triple Arthrodesis for Flatfoot Deformity: When, Why, and How?

Different types of arthrodesis for flatfoot deformity have a long history in foot and ankle surgery. Arthrodesis of the rearfoot can be a useful tool in helping correct deformity and maintaining that correction with good long-term results. Questions have risen recently however about the necessity of including the calcaneocuboid joint in the traditional rearfoot arthrodesis or triple arthrodesis. The double arthrodesis of the talonavicular and subtalar joints has grown in popularity and this review helps the reader choose with a review of the biomechanics, surgical approaches, fixation techniques and recent literature outcomes of both procedures.

Dexamethasone and OLT1177 Cooperate in the Reduction of Melanoma Growth by Inhibiting STAT3 Functions.

The NLRP3 inflammasome is a multimolecular complex that processes inactive IL-1ß and IL-18 into proinflammatory cytokines. OLT1177 is an orally active small compound that specifically inhibits NLRP3. Here, B16F10 melanoma were implanted in mice and treated with OLT1177 as well as combined with the glucocorticoid dexamethasone. At sacrifice, OLT1177 treated mice had significantly smaller tumors compared to tumor-bearing mice treated with vehicle. However, the combined treatment of OLT1177 plus dexamethasone revealed a greater suppression of tumor growth. This reduction was accompanied by a downregulation of nuclear and mitochondrial STAT3-dependent gene transcription and by a significant reduction of STAT3 Y705 and S727 phosphorylations in the tumors. In vitro, the human melanoma cell line 1205Lu, stimulated with IL-1α, exhibited significantly lower levels of STAT3 Y705 phosphorylation by the combination treatment, thus affecting the nuclear functions of STAT3. In the same cells, STAT3 serine 727 phosphorylation was also lower, affecting the mitochondrial functions of STAT3. In addition, metabolic analyses revealed a marked reduction of ATP production rate and glycolytic reserve in cells treated with the combination of OLT1177 plus dexamethasone. These findings demonstrate that the combination of OLT1177 and dexamethasone reduces tumor growth by targeting nuclear as well as mitochondrial functions of STAT3.

Metatarsal Protrusion Distance and Its Influence on Recurrent Ulceration Rates After Partial First-Ray Amputations: A Retrospective Study.

BACKGROUND: Recurrent ulceration is a common problem after partial first-ray amputations. Loss of the first metatarsophalangeal joint contributes to altered biomechanics and increased pressure on the foot. This may increase risk of adjacent ulcerations and additional amputations. Preserving first-ray length maintains the metatarsal parabola and limits transfer lesions, but few data support this. We aimed to evaluate the incidence of ulceration after partial first-ray amputations and to assess the association between metatarsal protrusion distance and recurrent ulceration. METHODS: Thirty-two consecutive patients underwent unilateral partial first-ray amputation at various levels along the first metatarsal, and the metatarsal protrusion distance was measured after surgery. Incidence of ulceration was evaluated on the ipsilateral foot. We hypothesized that patients with a longer first metatarsal were less likely to ulcerate again on the ipsilateral foot. RESULTS: Fourteen patients (43.8%) ulcerated again after partial first-ray amputation. Mean time to ulceration was 104 days. Active smoking status was associated with increased risk of another ulceration (P = .02), and chronic kidney disease was associated with a decreased risk of recurrent ulceration (P = .03). The average metatarsal protrusion distance for patients who ulcerated again after surgery was 36.1 mm versus 25.9 mm for patients who did not (P = .04). Logistic regression analysis of the receiver operating characteristic curve demonstrated an ideal cutoff length for recurrent ulceration of 37 mm (area under the curve = 0.7381). Patients with a protrusion distance greater than 37 mm were nine times as likely to ulcerate again (95% CI, 1.7-47.0). CONCLUSIONS: Partial first-ray amputations can be a good initial salvage procedure to clear infection and prolong bipedal ambulatory status. Unfortunately, these patients are prone to recurrent ulceration. Significant loss of first metatarsal length is a poor prognostic indicator for recurrent ulceration.

Activation of Host-NLRP3 Inflammasome in Myeloid Cells Dictates Response to Anti-PD-1 Therapy in Metastatic Breast Cancers.

Tumor-associated inflammation leads to dysregulated cytokine production that promotes tumor immune evasion and anti-tumor immunity dysfunction. In advanced stage breast cancer, the proinflammatory cytokine IL-1ß is overexpressed due to large proportions of activated myeloid cells in the tumor microenvironment (TME). Here, we demonstrate the role of the host nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing 3 (NLRP3) inflammasome in metastatic breast cancer. In vitro, we show that stimulation of THP-1 cells with conditioned media collected from MDA-MB-468 cells induced NLRP3 activation and increased Pdcd1l1 expression. In vivo, mice deficient in NLRP3 orthotopically implanted with metastatic breast cancer cell line (E0771) showed significant reduction in tumor growth (p < 0.05) and increased survival (p < 0.01). Inhibition of NLRP3 with the small molecule OLT1177® reduced expression of Pdcd1l1 (p < 0.001), Casp1 (p < 0.01) and Il1b (p < 0.01) in primary tumors. Furthermore, tumor-bearing mice receiving OLT1177® showed reduced infiltration of myeloid-derived suppressor cells (MDSCs) (p < 0.001) and increased CD8+ T cells (p < 0.05) and NK cells (p < 0.05) in the TME. NLRP3 inhibition in addition to anti-PD-1 treatment significantly reduced tumor growth from the monotherapies (p < 0.05). These data define NLRP3 activation as a key driver of immune suppression in metastatic breast cancers. Furthermore, this study suggests NLRP3 as a valid target to increase efficacy of immunotherapy with checkpoint inhibitor in metastatic breast cancers.

Langerhans cells and cDC1s play redundant roles in mRNA-LNP induced protective anti-influenza and anti-SARS-CoV-2 immune responses.

Nucleoside modified mRNA combined with Acuitas Therapeutics' lipid nanoparticles (LNPs) has been shown to support robust humoral immune responses in many preclinical animal vaccine studies and later in humans with the SARS-CoV-2 vaccination. We recently showed that this platform is highly inflammatory due to the LNPs' ionizable lipid component. The inflammatory property is key to support the development of potent humoral immune responses. However, the mechanism by which this platform drives T follicular helper (Tfh) cells and humoral immune responses remains unknown. Here we show that lack of Langerhans cells or cDC1s neither significantly affected the induction of PR8 HA and SARS-CoV-2 RBD-specific Tfh cells and humoral immune responses, nor susceptibility towards the lethal challenge of influenza and SARS-CoV-2. However, the combined deletion of these two DC subsets led to a significant decrease in the induction of PR8 HA and SARS-CoV-2 RBD-specific Tfh cell and humoral immune responses. Despite these observed defects, these mice remained protected from lethal influenza and SARS-CoV-2 challenges. We further found that IL-6, unlike neutrophils, was required to generate normal Tfh cells and antibody responses, but not for protection from influenza challenge. In summary, here we bring evidence that the mRNA-LNP platform can support the induction of protective immune responses in the absence of certain innate immune cells and cytokines.

Reulceration and Reoperation Incidence After Isolated Partial Fifth Ray Amputations: A Multicenter Study.

Ulceration or reulceration is a common complication following partial or total fifth ray amputations. The primary aim of this study was to evaluate the incidence of reulceration following partial fifth ray amputations. This was a multicenter review of 117 consecutive limbs that underwent partial fifth ray amputations at the University of Pittsburgh Medical Center and Wake Forest Baptist Medical Centers. Procedures were performed at various levels along the fifth metatarsal. Incidence of postoperative ulceration was evaluated on the ipsilateral foot. We hypothesized there would be an association between location of resection and development of reulceration. Seventy-one of 117 patients (60.7%) experienced repeat ulceration following a partial fifth ray amputation. Median follow-up time was 19 months. There was no statistical difference based on location of amputation (proximal, middle, distal, isolated base) with regards to reulceration (p = .166), further amputation (p = .271), transmetatarsal amputation (p = .160), or below knee amputation (p = .769). There was statistical significance in the follow up time between study sites (p = .013), fifth ray amputation reoperation rate between study sites (p = .001), and reulceration rates between study sites (p = .017). Partial fifth ray amputations can be a good initial salvage procedure to clear infection and prolong bipedal ambulatory status. The results of the present study put forward that there is not an association between location of amputations of the fifth ray and development of reulceration, transfer lesions or more proximal amputations.

Highly Potent SARS-CoV-2 Neutralization by Intravenous Immunoglobulins manufactured from Post-COVID-19 and COVID-19-Vaccinated Plasma Donations.

From September 2020, some immunoglobulin lots from US plasma contained neutralizing antibodies against the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Paralleled by the increasing numbers of post-coronavirus disease 2019 (COVID-19) donors, immunoglobulin lot antibody positivity increased to 93% by January 2021, at a mean titer of approximately 30 IU/mL. The correlation predicted that anti-SARS-CoV-2 potency would reach 345 IU/mL by July 2021. In addition to post-COVID-19 donors, the rapidly increasing number of plasma donors vaccinated against COVID-19 resulted in a mean antibody titer of >600 IU/mL in July 2021 immunoglobulin lots, with SARS-CoV-2 antibody titers for several lots even higher than those of earlier produced hyperimmune globulin products.

Tumor NLRP3-Derived IL-1ß Drives the IL-6/STAT3 Axis Resulting in Sustained MDSC-Mediated Immunosuppression.

Tumors evade the immune system by inducing inflammation. In melanoma, tumor-derived IL-1ß drives inflammation and the expansion of highly immunosuppressive myeloid-derived suppressor cells (MDSCs). Similar in many tumors, melanoma is also linked to the downstream IL-6/STAT3 axis. In this study, we observed that both recombinant and tumor-derived IL-1ß specifically induce pSTAT3(Y705), creating a tumor-autoinflammatory loop, which amplifies IL-6 signaling in the human melanoma cell line 1205Lu. To disrupt IL-1ß/IL-6/STAT3 axis, we suppressed IL-1ß-mediated inflammation by inhibiting the NOD-like receptor protein 3 (NLRP3) using OLT1177, a safe-in-humans specific NLRP3 oral inhibitor. In vivo, using B16F10 melanoma, OLT1177 effectively reduced tumor progression (p< 0.01); in primary tumors, OLT1177 decreased pSTAT3(Y705) by 82% (p<0.01) and II6 expression by 53% (p<0.05). Disruption of tumor-derived NLRP3, either pharmacologically or genetically, reduced STAT3 signaling in bone marrow cells. In PMN-MDSCs isolated from tumor-bearing mice treated with OLT1177, we observed significant reductions in immunosuppressive genes such as Pdcd1l1, Arg1, Il10 and Tgfb1. In conclusion, the data presented here show that the inhibition of NLRP3 reduces IL-1ß induction of pSTAT3(Y705) preventing expression of immunosuppressive genes as well as activity in PMN-MDSCs.

Outcomes of Tibiotalocalcaneal Arthrodesis with a Femoral Nail.

BACKGROUND: Retrograde intramedullary nailing for tibiotalocalcaneal arthrodesis (TTCA) is used for severe hindfoot deformities, end-stage arthritis, and limb salvage. The procedure is technically demanding, with complications such as infection, hardware failure, nonunion, osteomyelitis, and possible limb loss or death. This study reports the outcomes and complications of patients undergoing TTCA with a femoral nail, which is widely available and offers an extensive range of lengths and diameters. METHODS: We performed a retrospective review of 104 patients who underwent 109 TTCAs using a femoral nail as the primary procedure (January 2006 through December 2016). Demographic data, risk factors, and outcomes were evaluated. RESULTS: At final follow-up, the overall clinical union rate was 89 of 109 (81.7%). Diabetes mellitus was negatively associated with limb salvage (P = .03), and peripheral neuropathy (P = .02) and Charcot's neuroarthropathy (P = .03) were negatively associated with clinical union. Only four patients (3.8%) underwent proximal amputation, at an average of 6.1 months, and 11 patients (10.6%) died, at a mean of 38.0 months. The most common complication was ulceration in 27 of 109 limbs (24.8%), followed by infection in 25 (22.9%). Twenty-three patients (22.1%) underwent revision procedures, at a mean of 9.4 months. Thirteen of these 23 patients (56.5%) had antibiotic cement rod spacers/rods for deep infection-related complications. CONCLUSIONS: Use of a femoral nail has been shown to provide similar outcomes and limb salvage rates compared with other methods of TTCA reported for similar indications in the literature.

A novel anti-human IL-1R7 antibody reduces IL-18-mediated inflammatory signaling.

Unchecked inflammation can result in severe diseases with high mortality, such as macrophage activation syndrome (MAS). MAS and associated cytokine storms have been observed in COVID-19 patients exhibiting systemic hyperinflammation. Interleukin-18 (IL-18), a proinflammatory cytokine belonging to the IL-1 family, is elevated in both MAS and COVID-19 patients, and its level is known to correlate with the severity of COVID-19 symptoms. IL-18 binds its specific receptor IL-1 receptor 5 (IL-1R5, also known as IL-18 receptor alpha chain), leading to the recruitment of the coreceptor, IL-1 receptor 7 (IL-1R7, also known as IL-18 receptor beta chain). This heterotrimeric complex then initiates downstream signaling, resulting in systemic and local inflammation. Here, we developed a novel humanized monoclonal anti-IL-1R7 antibody to specifically block the activity of IL-18 and its inflammatory signaling. We characterized the function of this antibody in human cell lines, in freshly obtained peripheral blood mononuclear cells (PBMCs) and in human whole blood cultures. We found that the anti-IL-1R7 antibody significantly suppressed IL-18-mediated NFκB activation, reduced IL-18-stimulated IFNγ and IL-6 production in human cell lines, and reduced IL-18-induced IFNγ, IL-6, and TNFα production in PBMCs. Moreover, the anti-IL-1R7 antibody significantly inhibited LPS- and Candida albicans-induced IFNγ production in PBMCs, as well as LPS-induced IFNγ production in whole blood cultures. Our data suggest that blocking IL-1R7 could represent a potential therapeutic strategy to specifically modulate IL-18 signaling and may warrant further investigation into its clinical potential for treating IL-18-mediated diseases, including MAS and COVID-19.

Targeting tumor-derived NLRP3 reduces melanoma progression by limiting MDSCs expansion.

Interleukin-1ß (IL-1ß)-mediated inflammation suppresses antitumor immunity, leading to the generation of a tumor-permissive environment, tumor growth, and progression. Here, we demonstrate that nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation in melanoma is linked to IL-1ß production, inflammation, and immunosuppression. Analysis of cancer genome datasets (TCGA and GTEx) revealed greater NLRP3 and IL-1ß expression in cutaneous melanoma samples (n = 469) compared to normal skin (n = 324), with a highly significant correlation between NLRP3 and IL-1ß (P < 0.0001). We show the formation of the NLRP3 inflammasome in biopsies of metastatic melanoma using fluorescent resonance energy transfer analysis for NLRP3 and apoptosis-associated speck-like protein containing a CARD. In vivo, tumor-associated NLRP3/IL-1 signaling induced expansion of myeloid-derived suppressor cells (MDSCs), leading to reduced natural killer and CD8+ T cell activity concomitant with an increased presence of regulatory T (Treg) cells in the primary tumors. Either genetic or pharmacological inhibition of tumor-derived NLRP3 by dapansutrile (OLT1177) was sufficient to reduce MDSCs expansion and to enhance antitumor immunity, resulting in reduced tumor growth. Additionally, we observed that the combination of NLRP3 inhibition and anti-PD-1 treatment significantly increased the antitumor efficacy of the monotherapy by limiting MDSC-mediated T cell suppression and tumor progression. These data show that NLRP3 activation in melanoma cells is a protumor mechanism, which induces MDSCs expansion and immune evasion. We conclude that inhibition of NLRP3 can augment the efficacy of anti-PD-1 therapy.

Longitudinal analysis of SARS-CoV-2 antibodies in 8000 U.S. first-time convalescent plasma donations.

BACKGROUND: Coronavirus disease 2019 (COVID-19) convalescent individuals carry antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that, through a plasma donation, can be used as a potential therapeutic either in direct transfusion or for the manufacture of hyperimmune globulin (HIG). The success of such interventions depends on the antibody potency in such plasma donations, but little information on the collection of potent units is currently available. STUDY DESIGN AND METHODS: A total of 8749 plasma units, collected from April until September 2020 from first-time U.S. COVID-19 convalescent plasma donors, were characterized for SARS-CoV-2 immunoglobulin G (IgG) antibodies by Abbott chemiluminescent microparticle immunoassay (CMIA). The period between COVID-19 onset until donation and donor age, ethnicity, sex, and COVID-19 severity were evaluated against the obtained signal (index S/C). RESULTS: A marked decrease in mean index S/C was seen over the plasma collection period surveyed, which was significantly correlated to decreases in mean plasma donor age (p < .0001; R2 = .726) and percentage of donations obtained from COVID-19 convalescent patients who had been hospitalized (p = .001; R2 = .4426). The highest titer plasma units were obtained soon after convalescence from COVID-19 patients who required hospitalization, from advanced age donors, and from Black/African/Hispanic American versus White/Caucasian ethnicities, whereas there was no effect of donor sex on the values obtained with the Abbott CMIA. CONCLUSION: Since the onset of the pandemic, the average SARS-CoV-2 IgG values of first-time U.S. COVID-19 convalescent plasma donations have significantly dropped, mainly due to donations from progressively younger aged donors who tend to experience less severe COVID-19.

Human recombinant interleukin-38 suppresses inflammation in mouse models of local and systemic disease.

Interleukin (IL)-38 belongs to the IL-1 family and is part of the IL-36 subfamily due to its binding to the IL-36 Receptor (IL-1R6). In the current study, we assessed the anti-inflammatory properties of IL-38 in murine models of arthritis and systemic inflammation. First, the anti-inflammatory properties of mouse and human IL-38 precursors were compared to forms with a truncated N-terminus. In mouse bone marrow derived dendritic cells (BMDC), human and mouse IL-38 precursors with a truncation of the two N-terminal amino acids (3-152) suppressed LPS-induced IL-6. Recombinant human IL-38 (3-152) was further investigated for its immunomodulatory potential using four murine models of inflammatory disease: streptococcal cell wall (SCW)-induced arthritis, monosodium urate (MSU) crystal-induced arthritis, MSU crystal-induced peritonitis, and systemic endotoxemia. In each of these models IL-38 significantly reduced inflammation. In SCW and MSU crystal-induced arthritis, joint swelling, inflammatory cell influx, and synovial levels of IL-1ß, IL-6, and KC were reduced by 50% or greater. These suppressive properties of IL-38 in SCW-induced arthritis were independent of the anti-inflammatory co-receptor IL-1R8, as IL-38 reduced arthritis equally in IL-1R8 deficient and WT mice. In MSU crystal-induced peritonitis, IL-38 reduced hypothermia, while plasma IL-6 and KC and peritoneal KC levels were reduced by 65-70%. In the LPS endotoxemia model, IL-38 pretreatment reduced systemic IL-6, TNFα and KC. Furthermore, in ex vivo cultured bone marrow, LPS-induced IL-6, TNFα and KC were reduced by 75-90%. Overall, IL-38 exhibits broad anti-inflammatory properties in models of systemic and local inflammation and therefore may be an effective cytokine therapy.

Tibia Fracture in Staged Limb Salvage Using External Ring Fixation and Intramedullary Nailing: A Report of Two Cases.

Limb salvage for Charcot's neuroarthropathy has been shown to have high complication and failure rates. The aim of our report of two cases it to present a unique complication encountered with staged limb salvage for Charcot's neuroarthropathy. In two cases, patients developed delayed tibial shaft fracture associated with previous wire placement despite insertion of locked intramedullary nail fixation that spanned the delayed fracture. Both patients experienced fractures following advancement of weight after definitive fixation. In both patients, there was noted complication with the sites of the pins and revision of external fixation before fracture. In each case, the fracture was within the construct of the intramedullary fixation and successfully treated with an extended course of nonweightbearing. Complications of external fixation and intramedullary fixation are well reported within the literature; however, tibia fracture is rare. Based on these cases, it would seem prudent to recognize the risk of delayed pin-site complications and ensure adequate length of intramedullary fixation to span the potential areas of stress.

Metabolic Reprogramming of Mouse Bone Marrow Derived Macrophages Following Erythrophagocytosis.

Reticuloendothelial macrophages engulf ∼0.2 trillion senescent erythrocytes daily in a process called erythrophagocytosis (EP). This critical mechanism preserves systemic heme-iron homeostasis by regulating red blood cell (RBC) catabolism and iron recycling. Although extensive work has demonstrated the various effects on macrophage metabolic reprogramming by stimulation with proinflammatory cytokines, little is known about the impact of EP on the macrophage metabolome and proteome. Thus, we performed mass spectrometry-based metabolomics and proteomics analyses of mouse bone marrow-derived macrophages (BMDMs) before and after EP of IgG-coated RBCs. Further, metabolomics was performed on BMDMs incubated with free IgG to ensure that changes to macrophage metabolism were due to opsonized RBCs and not to free IgG binding. Uniformly labeled tracing experiments were conducted on BMDMs in the presence and absence of IgG-coated RBCs to assess the flux of glucose through the pentose phosphate pathway (PPP). In this study, we demonstrate that EP significantly alters amino acid and fatty acid metabolism, the Krebs cycle, OXPHOS, and arachidonate-linoleate metabolism. Increases in levels of amino acids, lipids and oxylipins, heme products, and RBC-derived proteins are noted in BMDMs following EP. Tracing experiments with U-13C6 glucose indicated a slower flux through glycolysis and enhanced PPP activation. Notably, we show that it is fueled by glucose derived from the macrophages themselves or from the extracellular media prior to EP, but not from opsonized RBCs. The PPP-derived NADPH can then fuel the oxidative burst, leading to the generation of reactive oxygen species necessary to promote digestion of phagocytosed RBC proteins via radical attack. Results were confirmed by redox proteomics experiments, demonstrating the oxidation of Cys152 and Cys94 of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hemoglobin-ß, respectively. Significant increases in early Krebs cycle and C5-branched dibasic acid metabolites (α-ketoglutarate and 2-hydroxyglutarate, respectively) indicate that EP promotes the dysregulation of mitochondrial metabolism. Lastly, EP stimulated aminolevulinic acid (ALA) synthase and arginase activity as indicated by significant accumulations of ALA and ornithine after IgG-mediated RBC ingestion. Importantly, EP-mediated metabolic reprogramming of BMDMs does not occur following exposure to IgG alone. In conclusion, we show that EP reprograms macrophage metabolism and modifies macrophage polarization.

Outcomes and Management of Infected Intramedullary Nails After Tibiotalocalcaneal Arthrodesis in Limb Salvage: A Retrospective Case Series.

Retrograde intramedullary nails are often used for tibiotalocalcaneal arthrodesis to correct severe hindfoot deformities in high-risk patient populations. The purposes of the current study are to report outcomes of patients undergoing staged management of infection after intramedullary nail fixation for tibiotalocalcaneal arthrodesis and to review the surgical approach to management of this limb-threatening complication. The authors reviewed patients who underwent hindfoot intramedullary nailing with subsequent revision for infection between January 2006 and December 2016. Staged protocol with antibiotic nail for the management of deep infection was used in 19 patients. The mean follow-up was 115.87 ± 92.80 (range 2.29 to 341.86) weeks. Twelve of the patients had diabetes, 10 had Charcot neuroarthropathy, and 7 had arthrodesis for equinovarus deformity. Sixteen had peripheral neuropathy and 13 had history of ulceration on the operated extremity. Limb salvage with the use of this protocol was achieved in 14 (73.68%) of 19 patients. Five (26.32%) patients had proximal amputation with 3 (15.79%) deaths within the follow-up period. Amputation was more likely in the nonsmoking (p = .01) and insulin-dependent (odds ratio = 22, p = .02) patient cohorts, whereas death was associated only with higher body mass index (p = .03). Time to revision was greater in patients with external bracing postoperatively as well (p = .004). Outcomes, including total number of procedures and retained antibiotic rods, were not associated with any of the preoperative variables or indications. In high-risk patient populations, the presented staged management of infected intramedullary hindfoot nails showed promising outcomes for limb preservation.

PASylation of IL-1 receptor antagonist (IL-1Ra) retains IL-1 blockade and extends its duration in mouse urate crystal-induced peritonitis.

Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS-IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS-IL-1Ra molecules, PAS600-IL-1Ra and PAS800-IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600-IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600-IL-1Ra and PAS800-IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800-IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800-IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600-IL-1Ra, and PAS800-IL-Ra reduced IL-1α-induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed Candida albicans-induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS-IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS-IL-1Ra as an active IL-1 antagonist with marked in vivo potency and a significantly extended half-life compared with anakinra.

Thermoeffector threshold plasticity: The impact of thermal pre-conditioning on sudomotor, cutaneous vasomotor and thermogenic thresholds.

To better understand the relationships between changes in body temperature and displacements of the thermoeffector thresholds (critical temperatures), the passive cooling (and heating) of pre-heated (and pre-cooled) individuals was investigated. Such experiments are necessary to understand the inter-dependence of those thresholds, and may possibly yield human evidence for the existence of separate central controllers. Eight males participated in four trials; two when normothermic, one following pre-experimental heating and the fourth following pre-cooling. Subjects were exposed to passive, whole-body cooling and heating when normothermic (the control trials), and again following pre-heating and pre-cooling (respectively). Cutaneous vasomotor, thermogenic, as well as precursor and discharged sudomotor thresholds from different body segments were compared across those dynamic thermal states. Following pre-heating, the critical mean body temperatures for vasoconstriction (0.37 °C ±â€¯0.10) and thermogenesis (0.67 °C ±â€¯0.20) were significantly elevated during passive cooling, relative to the corresponding control trial (both P < 0.05). When passive heating followed pre-cooling, the thresholds for vasodilatation were reduced (0.37 °C ±â€¯0.07; P < 0.05). Conversely, but with the exception of forehead precursor sweating, the sudomotor thresholds were elevated (averaging 0.16 °C ±â€¯0.02; P < 0.05). Most thermoeffectors revealed unique and adjustable activation thresholds, with the threshold displacements for thermogenesis and vasomotion appearing to be linked to the change in mean body temperature. Following pre-cooling, the critical temperatures for vasodilatation and sudomotor activation varied independently, with the exception of forehead precursor sweating. Collectively, those observations are consistent with the presence of independent central controllers for thermally dependent vasomotor and sudomotor responses, and perhaps also for shivering thermogenesis.

Evaluation of the safety and biodistribution of M032, an attenuated herpes simplex virus type 1 expressing hIL-12, after intracerebral administration to aotus nonhuman primates.

Herpes simplex virus type 1 (HSV-1) mutants lacking the γ(1)34.5 neurovirulence loci are promising agents for treating malignant glioma. Arming oncolytic HSV-1 to express immunostimulatory genes may potentiate therapeutic efficacy. We have previously demonstrated improved preclinical efficacy, biodistribution, and safety of M002, a γ(1)34.5-deleted HSV-1 engineered to express murine IL-12. Herein, we describe the safety and biodistribution of M032, a γ(1)34.5-deleted HSV-1 virus that expresses human IL-12 after intracerebral administration to nonhuman primates, Aotus nancymae. Cohorts were administered vehicle, 10(6), or 10(8) pfu of M032 on day 1 and subjected to detailed clinical observations performed serially over a 92-day trial. Animals were sacrificed on days 3, 31, and 91 for detailed histopathologic assessments of all organs and to isolate and quantify virus in all organs. With the possible exception of one animal euthanized on day 16, neither adverse clinical signs nor sex- or dose-related differences were attributed to M032. Elevated white blood cell and neutrophil counts were observed in virus-injected groups on day 3, but no other significant changes were noted in clinical chemistry or coagulation parameters. Minimal to mild inflammation and fibrosis detected, primarily in meningeal tissues, in M032-injected animals on days 3 and 31 had mostly resolved by day 91. The highest viral DNA levels were detected at the injection site and motor cortex on day 3 but decreased in central nervous system tissues over time. These data demonstrate the requisite safety of intracerebral M032 administration for consideration as a therapeutic for treating malignant brain tumors.

Transcriptome sequencing and development of an expression microarray platform for the domestic ferret.

BACKGROUND: The ferret (Mustela putorius furo) represents an attractive animal model for the study of respiratory diseases, including influenza. Despite its importance for biomedical research, the number of reagents for molecular and immunological analysis is restricted. We present here a parallel sequencing effort to produce an extensive EST (expressed sequence tags) dataset derived from a normalized ferret cDNA library made from mRNA from ferret blood, liver, lung, spleen and brain. RESULTS: We produced more than 500000 sequence reads that were assembled into 16000 partial ferret genes. These genes were combined with the available ferret sequences in the GenBank to develop a ferret specific microarray platform. Using this array, we detected tissue specific expression patterns which were confirmed by quantitative real time PCR assays. We also present a set of 41 ferret genes with even transcription profiles across the tested tissues, indicating their usefulness as housekeeping genes. CONCLUSION: The tools developed in this study allow for functional genomic analysis and make further development of reagents for the ferret model possible.