Systematic investigation of cytokine signaling activity at the tissue and single-cell levels.

Cytokines are critical for intercellular communication in human health and disease, but the investigation of cytokine signaling activity has remained challenging due to the short half-lives of cytokines and the complexity/redundancy of cytokine functions. To address these challenges, we developed the Cytokine Signaling Analyzer (CytoSig; https://cytosig.ccr.cancer.gov/ ), providing both a database of target genes modulated by cytokines and a predictive model of cytokine signaling cascades from transcriptomic profiles. We collected 20,591 transcriptome profiles for human cytokine, chemokine and growth factor responses. This atlas of transcriptional patterns induced by cytokines enabled the reliable prediction of signaling activities in distinct cell populations in infectious diseases, chronic inflammation and cancer using bulk and single-cell transcriptomic data. CytoSig revealed previously unidentified roles of many cytokines, such as BMP6 as an anti-inflammatory factor, and identified candidate therapeutic targets in human inflammatory diseases, such as CXCL8 for severe coronavirus disease 2019.

Intratumoral delivery of TransCon™ TLR7/8 Agonist promotes sustained anti-tumor activity and local immune cell activation while minimizing systemic cytokine induction.

BACKGROUND: Intratumoral (IT) delivery of toll-like receptor (TLR) agonists has shown encouraging anti-tumor benefit in preclinical and early clinical studies. However, IT delivery of TLR agonists may lead to rapid effusion from the tumor microenvironment (TME), potentially limiting the duration of local inflammation and increasing the risk of systemic adverse events. METHODS: To address these limitations, TransCon™ TLR7/8 Agonist-an investigational sustained-release prodrug of resiquimod that uses a TransCon linker and hydrogel technology to achieve sustained and predictable IT release of resiquimod-was developed. TransCon TLR7/8 Agonist was characterized for resiquimod release in vitro and in vivo, in mice and rats, and was assessed for anti-tumor efficacy and pharmacodynamic activity in mice. RESULTS: Following a single IT dose, TransCon TLR7/8 Agonist mediated potent tumor growth inhibition which was associated with sustained resiquimod release over several weeks with minimal induction of systemic cytokines. TransCon TLR7/8 Agonist monotherapy promoted activation of antigen-presenting cells in the TME and tumor-draining lymph nodes, with evidence of activation and expansion of CD8+ T cells in the tumor-draining lymph node and TME. Combination of TransCon TLR7/8 Agonist with systemic immunotherapy further promoted anti-tumor activity in TransCon TLR7/8 Agonist-treated tumors. In a bilateral tumor setting, combination of TransCon TLR7/8 Agonist with systemic IL-2 potentiated tumor growth inhibition in both injected and non-injected tumors and conferred protection against tumor rechallenge following complete regressions. CONCLUSIONS: Our findings show that a single dose of TransCon TLR7/8 Agonist can mediate sustained local release of resiquimod in the TME and promote potent anti-tumor effects as monotherapy and in combination with systemic immunotherapy, supporting TransCon TLR7/8 Agonist as a novel intratumoral TLR agonist for cancer therapy. A clinical trial to evaluate the safety and efficacy of TransCon TLR7/8 Agonist, as monotherapy and in combination with pembrolizumab, in cancer patients is currently ongoing (transcendIT-101; NCT04799054).

ShcA Protects against Epithelial-Mesenchymal Transition through Compartmentalized Inhibition of TGF-ß-Induced Smad Activation.

Epithelial-mesenchymal transition (EMT) is a normal cell differentiation event during development and contributes pathologically to carcinoma and fibrosis progression. EMT often associates with increased transforming growth factor-ß (TGF-ß) signaling, and TGF-ß drives EMT, in part through Smad-mediated reprogramming of gene expression. TGF-ß also activates the Erk MAPK pathway through recruitment and Tyr phosphorylation of the adaptor protein ShcA by the activated TGF-ß type I receptor. We found that ShcA protects the epithelial integrity of nontransformed cells against EMT by repressing TGF-ß-induced, Smad-mediated gene expression. p52ShcA competed with Smad3 for TGF-ß receptor binding, and down-regulation of ShcA expression enhanced autocrine TGF-ß/Smad signaling and target gene expression, whereas increased p52ShcA expression resulted in decreased Smad3 binding to the TGF-ß receptor, decreased Smad3 activation, and increased Erk MAPK and Akt signaling. Furthermore, p52ShcA sequestered TGF-ß receptor complexes to caveolin-associated membrane compartments, and reducing ShcA expression enhanced the receptor localization in clathrin-associated membrane compartments that enable Smad activation. Consequently, silencing ShcA expression induced EMT, with increased cell migration, invasion, and dissemination, and increased stem cell generation and mammosphere formation, dependent upon autocrine TGF-ß signaling. These findings position ShcA as a determinant of the epithelial phenotype by repressing TGF-ß-induced Smad activation through differential partitioning of receptor complexes at the cell surface.

Risk of Periprosthetic Fractures With Direct Anterior Primary Total Hip Arthroplasty.

BACKGROUND: Despite increasing interest in the anterior approach for cementless, primary total hip arthroplasty (THA), studies examining the incidence of periprosthetic fractures with this approach are lacking. The purpose of this study was to (1) investigate the incidence of early periprosthetic fractures associated with primary THA performed through an anterior supine intermuscular (ASI) approach without the use of a specialized table and (2) identify potential risk factors for these fractures. METHODS: We identified 2869 primary THAs performed via the ASI approach using a single cementless, tapered titanium femoral component with short and standard length options between February 2007 and April 2014. Fifty-two percent of THAs were in female patients, whereas 48% were in males. Short stems were used in 59% vs standard length in 41%. RESULTS: There were 26 (0.9%) early periprosthetic femoral fractures, with 23 requiring revision. When looking at the potential risk factors of age, gender, body mass index, and stem length, the only significant finding was that increased age was associated with increased risk of femoral fracture. Logistic regression analysis revealed a significant age-fracture association for female gender only, which remained when controlled for body mass index, stem length, or both. CONCLUSION: The muscle-sparing ASI approach appears to be a safe technique for performing primary THA when used in a suitable patient population. The early periprosthetic femoral fracture rate in our series may warrant consideration of using a different design or different approach in elderly female patients.

Frequency and Treatment Trends for Periprosthetic Fractures About Total Hip Arthroplasty in the United States.

BACKGROUND: Periprosthetic hip fractures (PPHFx) are challenging complications that have become increasingly more prevalent. Wide variability exists in the quality and size of prior studies pertaining to hospital stay information. This study used the largest publicly available database in the United States to evaluate perioperative hospital data of PPHFx. METHODS: The Healthcare Cost and Utilization Project-Nationwide Inpatient Sample was used to analyze trends related to the frequency, fracture type, mortality, treatment, patient demographics, time to surgery, length of stay (LOS), and hospital charges associated with PPHFx from 2006-2010. RESULTS: From 2006-2010, average patient age (76.7 years), hospital characteristics, rate of PPHFx, treatment choice, LOS (8.03 days), mortality (2.6%), disposition (78.1% to skilled nursing facility or inpatient rehab), and time to procedure (1.98 days) all remained relatively stable. The southern United States had the highest frequency of PPHFx and females had nearly twice the rate of PPHFx each year at an average of 67%. Despite these consistencies, hospital charges increased by an average of 8.3% per year over the study period ($27,683 over 5 years, P < .0001). CONCLUSION: In the era of containing cost while improving quality of care, this study demonstrates that despite consistent treatment trends of PPHFx, hospital charges are increasing independently. Regardless, surgeons can work to reduce LOS and charge to post acute care facilities to lessen spending. Refining our understanding of these relationships will be fundamental to further improving quality of care and cutting cost associated with these fractures.

Surgical fixation of extra-articular distal humerus fractures with a posterolateral plate through a triceps-reflecting technique.

BACKGROUND: Surgical management of extra-articular distal humerus fractures results in predictable fracture alignment. Open reduction and internal fixation also decrease the soft tissue complications and frequent follow-up required with functional bracing. A triceps-reflecting posterior approach provides excellent exposure to the humerus and minimizes trauma to the triceps. An anatomically precontoured plate on the posterolateral surface of the humerus provides stable fixation of these injuries and is placed directly through the interval developed by the triceps-reflecting approach. METHODS: We retrospectively reviewed the trauma databases at 2 level I academic trauma institutions during a 5-year period for all patients with an extra-articular distal humerus fracture treated with a triceps-reflecting approach and an anatomically precontoured posterolateral distal humerus plate. Patient and fracture characteristics were recorded, as were QuickDASH functional scores and visual analog scale scores for pain, function, and quality of life. RESULTS: Forty patients were eligible for our study. Average follow-up was 88 weeks. Thirty-eight (95%) patients went on to union. Seven (20%) patients required a secondary procedure. The average QuickDASH score was 17.5 (range, 2.6-56.8). The average visual analog scale scores were 1.9 (range, 0-7) for pain, 2.3 (range, 0-8) for function, and 1.6 (range, 0-5) for quality of life. Thirty-five (87.5%) patients reported satisfaction with the outcome of their surgery. DISCUSSION: Surgical fixation of extra-articular distal humerus fractures through a triceps-reflecting approach with an anatomically precontoured posterolateral distal humerus plate results in predictable osseous union and overall excellent functional results for patients with this injury.

Salvage hip arthroplasty after failed fixation of proximal femur fractures.

We reviewed 46 patients who underwent salvage hip arthroplasty (SHA) for revision of failed cannulated screws (CS), sliding hip screws (SHS), or intramedullary nails (IMN). The primary objective was to determine differences in operative difficulty. SHA after failed femoral neck fixation was associated with lower intra-operative demands than after failed peri-trochanteric fractures. Similarly, analysis by the index implant found that conversion arthroplasty after failed CSs was associated with lower intra-operative morbidity than failed SHSs or IMNs; differences between SHS and IMN were not as clear. Importantly, intra-operative data in cases of failed SHSs were similar regardless of the original fracture type, showing the device played a larger role than the fracture pattern. Complications and revision surgery rates were similar regardless of fracture type or fixation device. Our results suggest that operative demands and subsequent patient morbidity are more dependent on the index device than the fracture pattern during SHA.

Interleukin-1ß-regulating antibody XOMA 052 (gevokizumab) in the treatment of acute exacerbations of resistant uveitis of Behcet's disease: an open-label pilot study.

OBJECTIVE: Uveitis and retinal vasculitis are sight-threatening manifestations of Behçet's disease with limited treatment options. This pilot study aimed to evaluate the safety, pharmacokinetics and clinical activity of XOMA 052 (gevokizumab), a recombinant humanised anti-interleukin 1ß antibody, in Behçet's disease patients with uveitis. METHODS: Patients with acute posterior or panuveitis, and/or retinal vasculitis, resistant to azathioprine and/or ciclosporin, and receiving 10 mg/day or less of prednisolone, were enrolled into the 98-day study. Immunosuppressive agents were discontinued at baseline. Patients received a single infusion of XOMA 052 (0.3 mg/kg). The safety and uveitis status and pharmacokinetics of XOMA 052 were evaluated. RESULTS: Seven patients enrolled and completed the study. No treatment-related adverse event was observed. XOMA 052 treatment was associated with rapid and durable clinical response in all patients. Complete resolution of intraocular inflammation was achieved in 4-21 days (median 14 days), with a median duration of response of 49 days (range 21-97 days); one patient remained exacerbation free throughout the study. CONCLUSIONS: Well tolerated, XOMA 052 resulted in a rapid onset and sustained reduction in intraocular inflammation in patients with resistant uveitis and retinal vasculitis. Moreover, the effect was observed despite discontinuation of immunosuppressive agents and without the need to increase corticosteroid dosages.

Kinetic approach to pathway attenuation using XOMA 052, a regulatory therapeutic antibody that modulates interleukin-1beta activity.

Many therapeutic antibodies act as antagonists to competitively block cellular signaling pathways. We describe here an approach for the therapeutic use of monoclonal antibodies based on context-dependent attenuation to reduce pathologically high activity while allowing homeostatic signaling in biologically important pathways. Such attenuation is achieved by modulating the kinetics of a ligand binding to its various receptors and regulatory proteins rather than by complete blockade of signaling pathways. The anti-interleukin-1beta (IL-1beta) antibody XOMA 052 is a potent inhibitor of IL-1beta activity that reduces the affinity of IL-1beta for its signaling receptor and co-receptor but not for its decoy and soluble inhibitory receptors. This mechanism shifts the effective dose response of the cytokine so that the potency of IL-1beta bound by XOMA 052 is 20-100-fold lower than that of IL-1beta in the absence of antibody in a variety of in vitro cell-based assays. We propose that by decreasing potency of IL-1beta while allowing binding to its clearance and inhibitory receptors, XOMA 052 treatment will attenuate IL-1beta activity in concert with endogenous regulatory mechanisms. Furthermore, the ability to bind the decoy receptor may reduce the potential for accumulation of antibody.target complexes. Regulatory antibodies like XOMA 052, which selectively modulate signaling pathways, may represent a new mechanistic class of therapeutic antibodies.

Does Resident Participation in the Surgical Fixation of Hip Fractures Increase Operative Time or Affect Outcomes?

BACKGROUND: Surgical fixation of hip fractures is a common procedure at teaching hospitals with resident support and in community hospitals. OBJECTIVE: We evaluated to what extent participation by residents in hip fracture fixation affects operative times or outcomes. SETTING: Operations were performed by three surgeons who operate at a teaching hospital with resident support, and at a community hospital without residents in the same metropolitan area. PARTICIPANTS: We performed a retrospective analysis of operative time and early post-operative outcomes on a series of 314 patients with hip fractures (Arbeitsgemeinschaft für Osteosynthesefragen/Orthopaedic Trauma Association A1-3, B1-3) treated with surgical fixation between April 2012 and March 2015; 177 patients at the community hospital, and 137 at the teaching hospital. METHODS: Multivariate regression assessed the effect of hospital type, adjusting for age, gender, American Society of Anesthesiologist classification, and Charlson comorbidity index. RESULTS: We found lower median operative time at the community hospital than the teaching hospital (46 minutes, 95% confidence interval [CI] = [43, 52] versus 75 minutes, 95% CI = [70, 81]) and lower estimated blood loss (177.3 mL, 95% CI=[158.6, 195.1] versus 234.8 mL, 95% CI = [196.4, 273.6]), but no differences in transfusion requirement, length of stay, or discharge to skilled nursing facility. Adjusted odds ratio for thirty-day mortality at the teaching hospital was 5.44 (95% CI = [1.22, 24.1]). CONCLUSION: We found longer operative times and elevated estimated blood loss with resident involvement in surgical fixation of hip fractures. There was a difference in 30-day mortality between the groups, although this cannot simply be attributed to resident involvement as there are many other factors related to mortality.

Medical Comanagement of Hip Fracture Patients Is Not Associated with Superior Perioperative Outcomes: A Propensity Score-Matched Retrospective Cohort Analysis of the National Surgical Quality Improvement Project.

BACKGROUND: Medical comanagement entails a significant commitment of clinical resources with the aim of improving perioperative outcomes for patients admitted with hip fractures. To our knowledge, no national analyses have demonstrated whether patients benefit from this practice. METHODS: We performed a retrospective cohort analysis of the American College of Surgeons (ACS) National Surgical Quality Improvement Program (NSQIP) targeted user file for hip fracture 2016-2017. Medical comanagement is a dedicated variable in the NSQIP. Propensity score matching was performed to control for baseline differences associated with comanagement. Matched pairs binary logistic regression was then performed to determine the effect of comanagement on the following primary outcomes: mortality and a composite endpoint of major morbidity. RESULTS: Unadjusted analyses demonstrated that patients receiving medical comanagement were older and sicker with a greater burden of comorbidities. Comanagement did not have a higher proportion of patients participating in a standardized hip fracture program (53.6% vs 53.7%; P > .05). Comanagement was associated with a higher unadjusted rate of mortality (6.9% vs 4.0%, odds ratio [OR] 1.79: 1.44-2.22; P < .0001) and morbidity (19.5% vs 9.6%, OR 2.28: 1.98-2.63; P < .0001). After propensity score matching was used to control for baseline differences associated with comanagement, patients in the comanagement cohort continued to demonstrate inferior mortality (OR 1.36: 1.02-1.81; P = .033) and morbidity (OR 1.82: 1.52-2.20; P < .0001). CONCLUSIONS: This analysis does not provide evidence that dedicated medical comanagement of hip fracture patients is associated with superior perioperative outcomes. Further efforts may be needed to refine opportunities to modify the significant morbidity and mortality that persists in this population.

Case report: Patella baja after retrograde femoral nail insertion.

Patella baja is a rare condition that can result from conditions involving trauma around the knee. Risk factors are believed to include scar tissue formation in the retropatellar fat pad, extensor mechanism dysfunction, and immobilization in extension. Early recognition and aggressive treatment are critical components in minimizing long-term disability. We present a case report of a woman with a fracture of the femoral diaphysis who underwent retrograde placement of an intramedullary nail. Subsequent followup revealed development of patella baja with resultant disability. The diagnosis was made late and the treatment was ineffective. Although patella baja has been reported in trauma around the knee, causative factors include retrograde femoral nailing. We believe early recognition and institution of treatment are important.

Orthopaedic traumatology: fundamental principles and current controversies for the acute care surgeon.

Multiply injured patients with fractures are co-managed by acute care surgeons and orthopaedic surgeons. In most centers, orthopaedic surgeons definitively manage fractures, but preliminary management, including washouts, splinting, reductions, and external fixations, may be performed by selected acute care surgeons. The acute care surgeon should have a working knowledge of orthopaedic terminology to communicate with colleagues effectively. They should have an understanding of the composition of bone, periosteum, and cartilage, and their reaction when there is an injury. Fractures are usually fixed urgently, but some multiply injured patients are better served with a damage control strategy. Extremity compartment syndrome should be suspected in all critically injured patients with or without fractures and a low threshold for compartment pressure measurements or empiric fasciotomy maintained. Acute care surgeons performing rib fracture fixation and other chest wall injury reconstructions should follow the principles of open fracture reduction and stabilization.

Cooperative regulation of the cell division cycle by the protein kinases RAF and AKT.

The RAS-activated RAF-->MEK-->extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3'-kinase)-->PDK1-->AKT signaling pathways are believed to cooperate to promote the proliferation of normal cells and the aberrant proliferation of cancer cells. To explore the mechanisms that underlie such cooperation, we have derived cells harboring conditionally active, steroid hormone-regulated forms of RAF and AKT. These cells permit the assessment of the biological and biochemical effects of activation of these protein kinases either alone or in combination with one another. Under conditions where activation of neither RAF nor AKT alone promoted S-phase progression, coactivation of both kinases elicited a robust proliferative response. Moreover, under conditions where high-level activation of RAF induced G(1) cell cycle arrest, activation of AKT bypassed the arrest and promoted S-phase progression. At the level of the cell cycle machinery, RAF and AKT cooperated to induce cyclin D1 and repress p27(Kip1) expression. Repression of p27(Kip1) was accompanied by a dramatic reduction in KIP1 mRNA and was observed in primary mouse embryo fibroblasts derived from mice either lacking SKP2 or expressing a T187A mutated form of p27(Kip1). Consistent with these observations, pharmacological inhibition of MEK or PI3'-kinase inhibited the effects of activated RAS on the expression of p27(Kip1) in NIH 3T3 fibroblasts and in a panel of bona fide human pancreatic cancer cell lines. Furthermore, we demonstrated that AKT activation led to sustained activation of cyclin/cdk2 complexes that occurred concomitantly with the removal of RAF-induced p21(Cip1) from cyclin E/cdk2 complexes. Cumulatively, these data strongly suggest that the RAF-->MEK-->ERK and PI3'K-->PDK-->AKT signaling pathways can cooperate to promote G(0)-->G(1)-->S-phase cell cycle progression in both normal and cancer cells.

Blockade of only TGF-ß 1 and 2 is sufficient to enhance the efficacy of vaccine and PD-1 checkpoint blockade immunotherapy.

Checkpoint inhibition has established immunotherapy as a major modality of cancer treatment. However, the success of cancer immunotherapy is still limited as immune regulation of tumor immunity is very complicated and mechanisms involved may also differ among cancer types. Beside checkpoints, other good candidates for immunotherapy are immunosuppressive cytokines. TGF-ß is a very potent immunosuppressive cytokine involved in suppression of tumor immunity and also necessary for the function of some regulatory cells. TGF-ß has three isoforms, TGF-ß 1, 2 and 3. It has been demonstrated in multiple mouse tumor models that inhibition of all three isoforms of TGF-ß facilitates natural tumor immunosurveillance and tumor vaccine efficacy. However, individual isoforms of TGF-ß are not well studied yet. Here, by using monoclonal antibodies (mAbs) specific for TGF-ß isoforms, we asked whether it is necessary to inhibit TGF-ß3 to enhance tumor immunity. We found that blockade of TGF-ß1 and 2 and of all isoforms provided similar effects on tumor natural immunosurveillance and therapeutic vaccine-induced tumor immunity. The protection was CD8+ T cell-dependent. Blockade of TGF-ß increased vaccine-induced Th1-type response measured by IFNγ production or T-bet expression in both tumor draining lymph nodes and tumors, although it did not increase tumor antigen-specific CD8+ T cell numbers. Therefore, protection correlated with qualitative rather than quantitative changes in T cells. Furthermore, when combined with PD-1 blockade, blockade of TGF-ß1 and 2 further increased vaccine efficacy. In conclusion, blocking TGF-ß1 and 2 is sufficient to enhance tumor immunity, and it can be further enhanced with PD-1 blockade.

The Epidemiology of Primary and Revision Total Hip Arthroplasty in Teaching and Nonteaching Hospitals in the United States.

INTRODUCTION: The purpose of this study was to examine the epidemiology of primary and revision total hip arthroplasty (THA) in teaching and nonteaching hospitals. METHODS: The Healthcare Cost and Utilization Project Nationwide Inpatient Sample was queried from 2006 to 2010 to identify primary and revision THAs at teaching and nonteaching hospitals. RESULTS: A total of 1,336,396 primary and 223,520 revision procedures were identified. Forty-six percent of all primary and 54% of all revision procedures were performed at teaching hospitals. Teaching hospitals performed 17% of their THAs as revisions; nonteaching hospitals performed 12% as revisions. For primary and revision THAs, teaching hospitals had fewer patients aged >65 years, fewer Medicare patients, similar gender rates, more nonwhite patients, and more patients in the highest income quartile compared with nonteaching hospitals. Costs, length of stay, and Charlson Comorbidity Index scores were similar; however, the mortality rate was lower at teaching hospitals. CONCLUSIONS: This study found small but significant differences in key epidemiologic and outcome variables in examining primary and revision THA at teaching and nonteaching hospitals. LEVEL OF EVIDENCE: Level III.

Development and characterization of human monoclonal antibodies that neutralize multiple TGFß isoforms.

Transforming growth factor (TGF)ß levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFß1, 2, and 3. As multiple TGFß isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFß isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFß1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFß 1, 2 inhibition, but more modest affinity versus TGFß3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFß effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFß-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies' potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFß and may have utility for therapeutic intervention in human disease.

Quantitation of TGF-ß proteins in mouse tissues shows reciprocal changes in TGF-ß1 and TGF-ß3 in normal vs neoplastic mammary epithelium.

Transforming growth factor-ßs (TGF-ßs) regulate tissue homeostasis, and their expression is perturbed in many diseases. The three isoforms (TGF-ß1, -ß2, and -ß3) have similar bioactivities in vitro but show distinct activities in vivo. Little quantitative information exists for expression of TGF-ß isoform proteins in physiology or disease. We developed an optimized method to quantitate protein levels of the three isoforms, using a Luminex® xMAP®-based multianalyte assay following acid-ethanol extraction of tissues. Analysis of multiple tissues and plasma from four strains of adult mice showed that TGF-ß1 is the predominant isoform with TGF-ß2 being ~10-fold lower. There were no sex-specific differences in isoform expression, but some tissues showed inter-strain variation, particularly for TGF-ß2. The only adult tissue expressing appreciable TGF-ß3 was the mammary gland, where its levels were comparable to TGF-ß1. In situ hybridization showed the luminal epithelium as the major source of all TGF-ß isoforms in the normal mammary gland. TGF-ß1 protein was 3-8-fold higher in three murine mammary tumor models than in normal mammary gland, while TGF-ß3 protein was 2-3-fold lower in tumors than normal tissue, suggesting reciprocal regulation of these isoforms in mammary tumorigenesis.

Effects of the RAF/MEK/ERK and PI3K/AKT signal transduction pathways on the abrogation of cytokine-dependence and prevention of apoptosis in hematopoietic cells.

The Raf/MEK/ERK kinase cascade is pivotal in transmitting signals from membrane receptors to transcription factors that control gene expression culminating in the regulation of cell cycle progression. This cascade can prevent cell death through ERK2 and p90(Rsk) activation and phosphorylation of apoptotic and cell cycle regulatory proteins. The PI3K/Akt kinase cascade also controls apoptosis and can phosphorylate many apoptotic and cell cycle regulatory proteins. These pathways are interwoven as Akt can phosphorylate Raf and result in its inactivation, and Raf can be required for the antiapoptotic effects of Akt. In this study, the effects of activated Raf (Raf-1, A-Raf and B-Raf) and PI3K/Akt proteins on abrogation of cytokine dependence in FL5.12 hematopoietic cells were examined. Activated Raf, PI3K or Akt expression, by themselves, did not readily relieve cytokine dependence. The presence of activated Raf and PI3K/Akt increased the isolation of factor-independent cells from 400- to 2500-fold depending upon the particular combination examined. The individual effects of activated Raf and Akt on proliferation, apoptosis and autocrine growth factor synthesis were further examined with hormone-inducible constructs (Delta Raf-1:AR and Delta Akt:ER*(Myr(+)). Activation of either Raf or Akt hindered cell death; however, both proliferation and maximal synthesis of autocrine cytokines were dependent upon activation of both signaling pathways. The effects of small molecular weight inhibitors on DNA synthesis and cytokine gene expression were also examined. The PI3K inhibitor, LY294002, inhibited growth and cytokine gene expression. This effect could be synergistically increased by addition of the MEK inhibitor UO126. These cells will be useful in elucidating the interactions between Raf/MEK/ERK and PI3K/Akt cascades in proliferation, apoptosis, and leukemogenesis, as well as evaluating the efficacy of signal transduction inhibitors that target these cascades.

Initial management of pelvic and femoral fractures in the multiply injured patient.

The management of polytrauma patients is clinically challenging and requires a multi-disciplinary team approach. The immediate and definitive operative care of fractures represents the optimal treatment for polytrauma patients with orthopedic injuries. Early orthopedic intervention in long bone fractures and pelvic ring injuries has been shown to decrease pulmonary complications, improve hemodynamic stability, reduce ventilator time, and facilitate early patient mobilization. These factors decrease mortality and improve outcomes for patients with multiple injuries.