Exacerbated Staphylococcus aureus Foot Infections in Obese/Diabetic Mice Are Associated with Impaired Germinal Center Reactions, Ig Class Switching, and Humoral Immunity.

Obese patients with type 2 diabetes (T2D) are at an increased risk of foot infection, with impaired immune function believed to be a critical factor in the infectious process. In this study, we test the hypothesis that humoral immune defects contribute to exacerbated foot infection in a murine model of obesity/T2D. C57BL/6J mice were rendered obese and T2D by a high-fat diet for 3 mo and were compared with controls receiving a low-fat diet. Following injection of Staphylococcus aureus into the footpad, obese/T2D mice had greater foot swelling and reduced S. aureus clearance than controls. Obese/T2D mice also had impaired humoral immune responses as indicated by lower total IgG levels and lower anti-S. aureus Ab production. Within the draining popliteal lymph nodes of obese/T2D mice, germinal center formation was reduced, and the percentage of germinal center T and B cells was decreased by 40-50%. Activation of both T and B lymphocytes was similarly suppressed in obese/T2D mice. Impaired humoral immunity in obesity/T2D was independent of active S. aureus infection, as a similarly impaired humoral immune response was demonstrated when mice were administered an S. aureus digest. Isolated splenic B cells from obese/T2D mice activated normally but had markedly suppressed expression of Aicda, with diminished IgG and IgE responses. These results demonstrate impaired humoral immune responses in obesity/T2D, including B cell-specific defects in Ab production and class-switch recombination. Together, the defects in humoral immunity may contribute to the increased risk of foot infection in obese/T2D patients.

The Response of nor and nos Contributes to Staphylococcus aureus Virulence and Metabolism.

Staphylococcus aureus causes a wide spectrum of disease, with the site and severity of infection dependent on virulence traits encoded within genetically distinct clonal complexes (CCs) and bacterial responses to host innate immunity. The production of nitric oxide (NO) by activated phagocytes is a major host response to which S. aureus metabolically adapts through multiple strategies that are conserved in all CCs, including an S. aureus nitric oxide synthase (Nos). Previous genome analysis of CC30, a lineage associated with chronic endocardial and osteoarticular infections, revealed a putative NO reductase (Nor) not found in other CCs that potentially contributes to NO resistance and clinical outcome. Here, we demonstrate that Nor has true nitric oxide reductase activity, with nor expression enhanced by NO stress and anaerobic growth. Furthermore, we demonstrate that nor is regulated by MgrA and SrrAB, which modulate S. aureus virulence and hypoxic response. Transcriptome analysis of the S. aureus UAMS-1, UAMS-1 Δnor, and UAMS-1 Δnos strains under NO stress and anaerobic growth demonstrates that Nor contributes to nucleotide metabolism and Nos to glycolysis. We demonstrate that Nor and Nos contribute to enhanced survival in the presence of human human polymorphonuclear cells and have organ-specific seeding in a tail vein infection model. Nor contributes to abscess formation in an osteological implant model. We also demonstrate that Nor has a role in S. aureus metabolism and virulence. The regulation overlap between Nor and Nos points to an intriguing link between regulation of intracellular NO, metabolic adaptation, and persistence in the CC30 lineage.IMPORTANCEStaphylococcus aureus can cause disease at most body sites, and illness spans asymptomatic infection to death. The variety of clinical presentations is due to the diversity of strains, which are grouped into distinct clonal complexes (CCs) based on genetic differences. The ability of S. aureus CC30 to cause chronic infections relies on its ability to evade the oxidative/nitrosative defenses of the immune system and survive under different environmental conditions, including differences in oxygen and nitric oxide concentrations. The significance of this work is the exploration of unique genes involved in resisting NO stress and anoxia. A better understanding of the functions that control the response of S. aureus CC30 to NO and oxygen will guide the treatment of severe disease presentations.

Methionine Metabolites in Patients With Sepsis.

OBJECTIVE: Sepsis is characterized by microvascular dysfunction and thrombophilia. Several methionine metabolites may be relevant to this sepsis pathophysiology. S-adenosylmethionine (SAM) serves as the methyl donor for trans-methylation reactions. S-adenosylhomocysteine (SAH) is the by-product of these reactions and serves as the precursor to homocysteine. Relationships between plasma total homocysteine concentrations (tHcy) and vascular disease and thrombosis are firmly established. We hypothesized that SAM, SAH, and tHcy levels are elevated in patients with sepsis and associated with mortality. METHODS: This was a combined case-control and prospective cohort study consisting of 109 patients with sepsis and 50 control participants without acute illness. The study was conducted in the medical and surgical intensive care units of the University of Rochester Medical Center. Methionine, SAM, SAH, and tHcy concentrations were compared in patients with sepsis versus control participants and in sepsis survivors versus nonsurvivors. RESULTS: Patients with sepsis had significantly higher plasma SAM and SAH concentrations than control participants (SAM: 164 [107-227] vs73 [59-87 nM], P < .001; SAH: 99 [60-165] vs 35 [28-45] nM, P < .001). In contrast, plasma tHcy concentrations were lower in sepsis patients compared to healthy control participants (4 [2-6]) vs 7 [5-9] µM; P = .04). In multivariable analysis, quartiles of SAM, SAH, and tHcy were independently associated with sepsis ( P = .006, P = .05, and P < .001, respectively). Sepsis nonsurvivors had significantly higher plasma SAM and SAH concentrations than survivors (SAM: 223 [125-260] vs 136 [96-187] nM; P = .01; SAH: 139 [81-197] vs 86 [55-130] nM, P = .006). Plasma tHcy levels were similar in survivors vs nonsurvivors. The associations between SAM or SAH and hospital mortality were no longer significant after adjusting for renal dysfunction. CONCLUSIONS: Methionine metabolite concentrations are abnormal in sepsis and linked with clinical outcomes. Further study is required to determine whether these abnormalities have pathophysiologic significance.

Adaptive Upregulation of Clumping Factor A (ClfA) by Staphylococcus aureus in the Obese, Type 2 Diabetic Host Mediates Increased Virulence.

Obesity and associated type 2 diabetes (T2D) are important risk factors for infection following orthopedic implant surgery. Staphylococcus aureus, the most common pathogen in bone infections, adapts to multiple environments to survive and evade host immune responses. Whether adaptation of S. aureus to the unique environment of the obese/T2D host accounts for its increased virulence and persistence in this population is unknown. Thus, we assessed implant-associated osteomyelitis in normal versus high-fat-diet obese/T2D mice and found that S. aureus infection was more severe, including increases in bone abscesses relative to nondiabetic controls. S. aureus isolated from bone of obese/T2D mice displayed marked upregulation of four adhesion genes (clfA, clfB, bbp, and sdrC), all with binding affinity for fibrin(ogen). Immunostaining of infected bone revealed increased fibrin deposition surrounding bacterial abscesses in obese/T2D mice. In vitro coagulation assays demonstrated a hypercoagulable state in obese/T2D mice that was comparable to that of diabetic patients. S. aureus with an inactivating mutation in clumping factor A (clfA) showed a reduction in bone infection severity that eliminated the effect of obesity/T2D, while infections in control mice were unchanged. In infected mice that overexpress plasminogen activator inhibitor-1 (PAI-1), S. aureusclfA expression and fibrin-encapsulated abscess communities in bone were also increased, further linking fibrin deposition to S. aureus expression of clfA and infection severity. Together, these results demonstrate an adaptation by S. aureus to obesity/T2D with increased expression of clfA that is associated with the hypercoagulable state of the host and increased virulence of S. aureus.

A humoral immune defect distinguishes the response to Staphylococcus aureus infections in mice with obesity and type 2 diabetes from that in mice with type 1 diabetes.

Obesity and diabetes are among the greatest risk factors for infection following total joint arthroplasty. However, the underlying mechanism of susceptibility is unclear. We compared orthopedic implant-associated Staphylococcus aureus infections in type 1 (T1D) versus type 2 (T2D) diabetic mouse models and in patients with S. aureus infections, focusing on the adaptive immune response. Mice were fed a high-fat diet to initiate obesity and T2D. T1D was initiated with streptozotocin. Mice were then given a trans-tibial implant that was precoated with bioluminescent Xen36 S. aureus. Although both mouse models of diabetes demonstrated worse infection severity than controls, infection in T2D mice was more severe, as indicated by increases in bioluminescence, S. aureus CFU in tissue, and death within the first 7 days. Furthermore, T2D mice had an impaired humoral immune response at day 14 with reduced total IgG, decreased S. aureus-specific IgG, and increased IgM. These changes were not present in T1D mice. Similarly, T2D patients and obese nondiabetics with active S. aureus infections had a blunted IgG response to S. aureus. In conclusion, we report the first evidence of a humoral immune deficit, possibly due to an immunoglobulin class switch defect, in obesity and T2D during exacerbated S. aureus infection which may contribute to the increased infection risk following arthroplasty in patients with T2D and obesity.

High-fat diet causes bone loss in young mice by promoting osteoclastogenesis through alteration of the bone marrow environment.

Obesity is a severe health problem in children, afflicting several organ systems including bone. However, the role of obesity on bone homeostasis and bone cell function in children has not been studied in detail. Here we used young mice fed a high-fat diet (HFD) to model childhood obesity and investigate the effect of HFD on the phenotype of cells within the bone marrow environment. Five-week-old male mice were fed a HFD for 3, 6, and 12 weeks. Decreased bone volume was detected after 3 weeks of HFD treatment. After 6 and 12 weeks, HFD-exposed mice had less bone mass and increased osteoclast numbers. Bone marrow cells, but not spleen cells, from HFD-fed mice had increased osteoclast precursor frequency, elevated osteoclast formation, and bone resorption activity, as well as increased expression of osteoclastogenic regulators including RANKL, TNF, and PPAR-gamma. Bone formation rate and osteoblast and adipocyte numbers were also increased in HFD-fed mice. Isolated bone marrow cells also had a corresponding elevation in the expression of positive regulators of osteoblast and adipocyte differentiation. Our findings indicate that in juvenile mice, HFD-induced bone loss is mainly due to increased osteoclast bone resorption by affecting the bone marrow microenvironment. Thus, targeting osteoclast formation may present a new therapeutic approach for bone complications in obese children.

Shear wave dispersion in lean versus steatotic rat livers.

OBJECTIVES: The precise measurement of fat accumulation in the liver, or steatosis, is an important clinical goal. Our previous studies in phantoms and mouse livers support the hypothesis that, starting with a normal liver, increasing accumulations of microsteatosis and macrosteatosis will increase the lossy viscoelastic properties of shear waves in a medium. This increase results in an increased dispersion (or slope) of the shear wave speed in the steatotic livers. METHODS: In this study, we moved to a larger animal model, lean versus obese rat livers ex vivo, and a higher-frequency imaging system to estimate the shear wave speed from crawling waves. RESULTS: The results showed elevated dispersion in the obese rats and a separation of the lean versus obese liver parameters in a 2-dimensional parameter space of the dispersion (slope) and shear wave speed at a reference frequency of 150 Hz. CONCLUSIONS: We have confirmed in 3 separate studies the validity of our dispersion hypothesis in animal models.

RBP-Jκ-dependent Notch signaling is required for murine articular cartilage and joint maintenance.

OBJECTIVE: Osteoarthritis (OA) is a degenerative disease resulting in severe joint cartilage destruction and disability. While the mechanisms underlying the development and progression of OA are poorly understood, gene mutations have been identified within cartilage-related signaling molecules, implicating impaired cell signaling in OA and joint disease. The Notch pathway has recently been identified as a crucial regulator of growth plate cartilage development, and components are expressed in joint tissue. This study was undertaken to investigate a novel role for Notch signaling in joint cartilage development, maintenance, and the pathogenesis of joint disease in a mouse model. METHODS: We performed the first mouse gene study in which the core Notch signaling component, RBP-Jκ, was tissue specifically deleted within joints. The Prx1Cre transgene removed Rbpjk loxP-flanked alleles in mesenchymal joint precursor cells, while the Col2Cre(ERT2) transgene specifically deleted Rbpjk in postnatal chondrocytes. Murine articular chondrocyte cultures were also used to examine Notch regulation of gene expression. RESULTS: Loss of Notch signaling in mesenchymal joint precursor cells did not affect embryonic joint development in mice, but rather, resulted in an early, progressive OA-like pathology. Additionally, partial loss of Notch signaling in murine postnatal cartilage resulted in progressive joint cartilage degeneration and an age-related OA-like pathology. Inhibition of Notch signaling altered the expression of the extracellular matrix (ECM)-related factors type II collagen (COL2A1), proteoglycan 4, COL10A1, matrix metalloproteinase 13, and ADAMTS. CONCLUSION: Our findings indicate that the RBP-Jκ-dependent Notch pathway is a novel pathway involved in joint maintenance and articular cartilage homeostasis, a critical regulator of articular cartilage ECM-related molecules, and a potentially important therapeutic target for OA-like joint disease.

Midtrimester fetal herpes simplex-2 diagnosis by serology, culture and quantitative polymerase chain reaction.

The acquisition of herpes simplex virus (HSV) in utero comprises a minority of neonatal herpes infections. Prenatal diagnosis is rare. We describe a midtrimester diagnosis of fetal HSV-2 infection. Ultrasound at 20 weeks for elevated maternal serum α-fetoprotein (MSAFP) showed lagging fetal growth, echogenic bowel, echogenic myocardium, and liver with a mottled pattern of echogenicity. Amniocentesis demonstrated normal karyotype, elevated AFP and positive acetylcholinesterase. Culture isolated HSV-2 with an aberrant growth pattern. Maternal serology was positive for HSV-2. Quantitative DNA polymerase chain reaction (PCR) showed 59 million copies/ml. Fetal autopsy demonstrated widespread tissue necrosis but only sparse HSV-2 inclusions. Fetal HSV-2 infection can be suspected when an elevated MSAFP accompanies ultrasound findings suggesting perinatal infection. Maternal HSV serology, amniotic fluid culture and quantitative PCR are recommended for diagnostic certainty and counseling.

Modulation of Gut Microbiota Metabolism in Obesity-Related Type 2 Diabetes Reduces Osteomyelitis Severity.

Staphylococcus aureus is an opportunistic pathogen causing osteomyelitis through hematogenous seeding or contamination of implants and open wounds following orthopedic surgeries. The severity of S. aureus-mediated osteomyelitis is enhanced in obesity-related type 2 diabetes (obesity/T2D) due to chronic inflammation impairing both adaptive and innate immunity. Obesity-induced inflammation is linked to gut dysbiosis, with modification of the gut microbiota by high-fiber diets leading to a reduction in the symptoms and complications of obesity/T2D. However, our understanding of the mechanisms by which modifications of the gut microbiota alter host infection responses is limited. To address this gap, we monitored tibial S. aureus infections in obese/T2D mice treated with the inulin-like fructan fiber oligofructose. Treatment with oligofructose significantly decreased S. aureus colonization and lowered proinflammatory signaling postinfection in obese/T2D mice, as observed by decreased circulating inflammatory cytokines (tumor necrosis factor-α [TNF-α]) and chemokines (interferon-γ-induced protein 10 kDa [IP-10], keratinocyte-derived chemokine [KC], monokine induced by interferon-γ [MIG], monocyte chemoattractant protein-1 [MCP-1], and regulated upon activation, normal T cell expressed and presumably secreted [RANTES]), indicating partial reduction in inflammation. Oligofructose markedly shifted diversity in the gut microbiota of obese/T2D mice, with notable increases in the anti-inflammatory bacterium Bifidobacterium pseudolongum. Analysis of the cecum and plasma metabolome suggested that polyamine production was increased, specifically spermine and spermidine. Oral administration of these polyamines to obese/T2D mice resulted in reduced infection severity similar to oligofructose supplementation, suggesting that polyamines can mediate the beneficial effects of fiber on osteomyelitis severity. These results demonstrate the contribution of gut microbiota metabolites to the control of bacterial infections distal to the gut and polyamines as an adjunct therapeutic for osteomyelitis in obesity/T2D. IMPORTANCE Individuals with obesity-related type 2 diabetes (obesity/T2D) are at a five times increased risk for invasive Staphylococcus aureus osteomyelitis (bone infection) following orthopedic surgeries. With increasing antibiotic resistance and limited discoveries of novel antibiotics, it is imperative that we explore other avenues for therapeutics. In this study, we demonstrated that the dietary fiber oligofructose markedly reduced osteomyelitis severity and hyperinflammation following acute prosthetic joint infections in obese/T2D mice. Reduced infection severity was associated with changes in gut microbiota composition and metabolism, as indicated by increased production of natural polyamines in the gut and circulating plasma. This work identifies a novel role for the gut microbiome in mediating control of bacterial infections and polyamines as beneficial metabolites involved in improving the obesity/T2D host response to osteomyelitis. Understanding the impact of polyamines on host immunity and mechanisms behind decreasing susceptibility to severe implant-associated osteomyelitis is crucial to improving treatment strategies for this patient population.

Suppressor of cytokine signaling-3 is a glucagon-inducible inhibitor of PKA activity and gluconeogenic gene expression in hepatocytes.

SOCS3 is a cytokine-inducible negative regulator of cytokine receptor signaling. Recently, SOCS3 was shown to be induced by a cAMP-dependent pathway involving exchange protein directly activated by cAMP (Epac). We observed in livers of fasted mice that Socs3 mRNA was increased 4-fold compared with refed mice, suggesting a physiologic role for SOCS3 in the fasted state that may involve glucagon and Epac. Treating primary hepatocytes with glucagon resulted in a 4-fold increase in Socs3 mRNA levels. The Epac-selective cAMP analog 8-4-(chlorophenylthio)-2'-O-methyladenosine-3',5'-monophosphate, acetoxymethyl ester (cpTOME) increased Socs3 expression comparably. In gain-of-function studies, adenoviral expression of SOCS3 in primary hepatocytes caused a 50% decrease in 8-br-cAMP-dependent PKA phosphorylation of the transcription factor CREB. Induction of the gluconeogenic genes Ppargc1a, Pck1, and G6pc by glucagon or 8-br-cAMP was suppressed nearly 50%. In loss-of-function studies, hepatocytes from liver-specific SOCS3 knock-out mice responded to 8-br-cAMP with a 200% greater increase in Ppargc1a and Pck1 expression, and a 30% increase in G6pc expression, relative to wild-type cells. Suppression of SOCS3 by shRNA in hepatocytes resulted in a 60% increase in cAMP-dependent G6pc and Pck1 expression relative to control cells. SOCS3 expression also inhibited cAMP-dependent phosphorylation of the IP3 receptor but did not inhibit nuclear localization of the catalytic subunit of PKA. Using an in vitro kinase assay, cAMP-dependent PKA activity was reduced by 80% in hepatocytes expressing ectopic SOCS3. These data indicate that cAMP activates both the PKA and Epac pathways with induction of SOCS3 by the Epac pathway negatively regulating the PKA pathway.

Metabolic regulation of APOBEC-1 complementation factor trafficking in mouse models of obesity and its positive correlation with the expression of ApoB protein in hepatocytes.

APOBEC-1 Complementation Factor (ACF) is an RNA-binding protein that interacts with apoB mRNA to support RNA editing. ACF traffics between the cytoplasm and nucleus. It is retained in the nucleus in response to elevated serum insulin levels where it supports enhanced apoB mRNA editing. In this report we tested whether ACF may have the ability to regulate nuclear export of apoB mRNA to the sites of translation in the cytoplasm. Using mouse models of obesity-induced insulin resistance and primary hepatocyte cultures we demonstrated that both nuclear retention of ACF and apoB mRNA editing were reduced in the livers of hyperinsulinemic obese mice relative to lean controls. Coincident with an increase in the recovery of ACF in the cytoplasm was an increase in the proportion of total cellular apoB mRNA recovered in cytoplasmic extracts. Cytoplasmic ACF from both lean controls and obese mouse livers was enriched in endosomal fractions associated with apoB mRNA translation and ApoB lipoprotein assembly. Inhibition of ACF export to the cytoplasm resulted in nuclear retention of apoB mRNA and reduced both intracellular and secreted ApoB protein in primary hepatocytes. The importance of ACF for modulating ApoB was supported by the finding that RNAi knockdown of ACF reduced ApoB secretion. An additional discovery from this study was the finding that leptin is a suppressor ACF expression. Dyslipidemia is a common pathology associated with insulin resistance that is in part due to the loss of insulin controlled secretion of lipid in ApoB-containing very low density lipoproteins. The data from animal models suggested that loss of insulin regulated ACF trafficking and leptin regulated ACF expression may make an early contribution to the overall pathology associated with very low density lipoprotein secretion from the liver in obese individuals.

The ratio of arginine to dimethylarginines is reduced and predicts outcomes in patients with severe sepsis.

OBJECTIVES: Arginine deficiency may contribute to microvascular dysfunction, but previous studies suggest that arginine supplementation may be harmful in sepsis. Systemic arginine availability can be estimated by measuring the ratio of arginine to its endogenous inhibitors, asymmetric and symmetric dimethylarginine. We hypothesized that the arginine-to-dimethylarginine ratio is reduced in patients with severe sepsis and associated with severity of illness and outcomes. DESIGN: Case-control and prospective cohort study. SETTING: Medical and surgical intensive care units of an academic medical center. PATIENTS AND SUBJECTS: One hundred nine severe sepsis and 50 control subjects. MEASUREMENTS AND MAIN RESULTS: Plasma and urine were obtained in control subjects and within 48 hrs of diagnosis in severe sepsis patients. The arginine-to-dimethylarginine ratio was higher in control subjects vs. sepsis patients (median, 95; interquartile range, 85-114; vs. median, 34; interquartile range, 24-48; p < .001) and in hospital survivors vs. nonsurvivors (median, 39; interquartile range, 26-52; vs. median, 27; interquartile range, 19-32; p = .004). The arginine-to-dimethylarginine ratio was correlated with Acute Physiology and Chronic Health Evaluation II score (Spearman's correlation coefficient [ρ] = - 0.40; p < .001) and organ-failure free days (ρ = 0.30; p = .001). A declining arginine-to-dimethylarginine ratio was independently associated with hospital mortality (odds ratio, 1.63 per quartile; 95% confidence interval, 1.00-2.65; p = .048) and risk of death over the course of 6 months (hazard ratio, 1.41 per quartile; 95% confidence interval, 1.01-1.98; p = .043). The arginine-to-dimethylarginine ratio was correlated with the urinary nitrate-to-creatinine ratio (ρ = 0.46; p < .001). CONCLUSIONS: The arginine-to-dimethylarginine ratio is associated with severe sepsis, severity of illness, and clinical outcomes. The arginine-to-dimethylarginine ratio may be a useful biomarker, and interventions designed to augment systemic arginine availability in severe sepsis may still be worthy of investigation.

Loss of Kupffer cells in diet-induced obesity is associated with increased hepatic steatosis, STAT3 signaling, and further decreases in insulin signaling.

While adipose tissue-associated macrophages contribute to development of chronic inflammation and insulin resistance of obesity, little is known about the role of hepatic Kupffer cells in this environment. Here we address the impact of Kupffer cell ablation using clodronate-encapsulated liposome depletion in a diet-induced obese (DIO) and insulin resistant mouse model. Hepatic expression of macrophage markers measured by realtime RT-PCR remained unaltered in DIO mice despite characteristic expansion of adipose tissue-associated macrophages. DIO mouse livers displayed increased expression of alternative activation markers but unaltered proinflammatory cytokine expression when compared to lean mice. Kupffer cell ablation reduced hepatic anti-inflammatory cytokine IL-10 mRNA expression in lean and DIO mice by 95% and 84%, respectively. Despite decreased hepatic IL-6 gene expression after ablation in lean and DIO mice, hepatic STAT3 phosphorylation, Socs3 and acute phase protein mRNA expression increased. Kupffer cell ablation in DIO mice resulted in additional hepatic triglyceride accumulation and a 30-40% reduction in hepatic insulin receptor autophosphorylation and Akt activation. Implicating systemic loss of IL-10, high-fat-fed IL-10 knockout mice also displayed increased hepatic STAT3 signaling and hepatic triglyceride accumulation. Insulin signaling was not altered, however. In conclusion, Kupffer cells are a major source of hepatic IL-10 expression, the loss of which is associated with increased STAT3-dependent signaling and steatosis. One or more additional factors appear to be required, however, for the Kupffer cell-dependent protective effect on insulin receptor signaling in DIO mice.

Recommendations for design and conduct of preclinical in vivo studies of orthopedic device-related infection.

Orthopedic device-related infection (ODRI), including both fracture-related infection (FRI) and periprosthetic joint infection (PJI), remain among the most challenging complications in orthopedic and musculoskeletal trauma surgery. ODRI has been convincingly shown to delay healing, worsen functional outcome and incur significant socio-economic costs. To address this clinical problem, ever more sophisticated technologies targeting the prevention and/or treatment of ODRI are being developed and tested in vitro and in vivo. Among the most commonly described innovations are antimicrobial-coated orthopedic devices, antimicrobial-loaded bone cements and void fillers, and dual osteo-inductive/antimicrobial biomaterials. Unfortunately, translation of these technologies to the clinic has been limited, at least partially due to the challenging and still evolving regulatory environment for antimicrobial drug-device combination products, and a lack of clarity in the burden of proof required in preclinical studies. Preclinical in vivo testing (i.e. animal studies) represents a critical phase of the multidisciplinary effort to design, produce and reliably test both safety and efficacy of any new antimicrobial device. Nonetheless, current in vivo testing protocols, procedures, models, and assessments are highly disparate, irregularly conducted and reported, and without standardization and validation. The purpose of the present opinion piece is to discuss best practices in preclinical in vivo testing of antimicrobial interventions targeting ODRI. By sharing these experience-driven views, we aim to aid others in conducting such studies both for fundamental biomedical research, but also for regulatory and clinical evaluation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:271-287, 2019.

Maternal serum alpha-fetoprotein values in type 1 and type 2 diabetic patients.

OBJECTIVE: Maternal serum alpha-fetoprotein (MSAFP) values are reported to be lower in type 1 diabetic patients, and a correction factor is often applied. We sought to determine whether type 2 diabetic patients require the same MSAFP adjustments as type 1 diabetic patients. STUDY DESIGN: We performed a retrospective review of MSAFP levels from a university laboratory in type 1 and type 2 diabetic patients between July 2000 and August 2006, matched 1:2 with controls. Groups were compared using analysis of variance and Student t testing. RESULTS: Seventy-seven type 1 and 75 type 2 diabetic patients were compared with 304 controls. Type 1 and type 2 diabetic patients differed significantly from each other and controls before corrections. Diabetic patients were similar to each other, but significantly lower than controls, after weight corrections. These differences were eliminated by a 10% correction factor. CONCLUSION: Type 1 and type 2 diabetic patients require both weight and diabetes corrections to adjust MSAFP values to nondiabetic levels.

Obesity/type 2 diabetes increases inflammation, periosteal reactive bone formation, and osteolysis during Staphylococcus aureus implant-associated bone infection.

Obese and type 2 diabetic (T2D) patients have a fivefold increased rate of infection following placement of an indwelling orthopaedic device. Though implant infections are associated with inflammation, periosteal reactive bone formation, and osteolysis, the effect of obesity/T2D on these complicating factors has not been studied. To address this question, C57BL/6J mice were fed a high fat diet (60% Kcal from fat) to induce obesity/T2D, or a control diet (10% Kcal from fat) for 3 months, and challenged with a transtibial pin coated with a bioluminescent USA300 strain of S. aureus. In the resulting infected bone, obesity/T2D was associated with increased S. aureus proliferation and colony forming units. RNA sequencing of the infected tibiae on days 7 and 14 revealed an increase in 635 genes in obese/T2D mice relative to controls. Pathways associated with ossification, angiogenesis, and immunity were enriched. MicroCT and histology on days 21 and 35 demonstrated significant increased periosteal reactive bone formation in infected obese/T2D mice versus infected controls (p < 0.05). The enhanced periosteal bone formation was associated with increased osteoblastic activity and robust endochondral ossification, with persistant cartilage on day 21 that was only observed in infected obesity/T2D. Osteolysis and osteoclast numbers in obesity/T2D were also significantly increased versus infected controls (p < 0.05). Consistent with an up-regulated immune transcriptome, macrophages were more abundant within both the periosteum and the new reactive bone of obese/T2D mice. In conclusion, we find that implant-associated S. aureus osteomyelitis in obesity/T2D is associated with increased inflammation, reactive bone formation, and osteolysis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1614-1623, 2018.

Targeting the gut microbiome to treat the osteoarthritis of obesity.

Obesity is a risk factor for osteoarthritis (OA), the greatest cause of disability in the US. The impact of obesity on OA is driven by systemic inflammation, and increased systemic inflammation is now understood to be caused by gut microbiome dysbiosis. Oligofructose, a nondigestible prebiotic fiber, can restore a lean gut microbial community profile in the context of obesity, suggesting a potentially novel approach to treat the OA of obesity. Here, we report that - compared with the lean murine gut - obesity is associated with loss of beneficial Bifidobacteria, while key proinflammatory species gain in abundance. A downstream systemic inflammatory signature culminates with macrophage migration to the synovium and accelerated knee OA. Oligofructose supplementation restores the lean gut microbiome in obese mice, in part, by supporting key commensal microflora, particularly Bifidobacterium pseudolongum. This is associated with reduced inflammation in the colon, circulation, and knee and protection from OA. This observation of a gut microbiome-OA connection sets the stage for discovery of potentially new OA therapeutics involving strategic manipulation of specific microbial species inhabiting the intestinal space.

Daily oral consumption of hydrolyzed type 1 collagen is chondroprotective and anti-inflammatory in murine posttraumatic osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease for which there are no disease modifying therapies. Thus, strategies that offer chondroprotective or regenerative capability represent a critical unmet need. Recently, oral consumption of a hydrolyzed type 1 collagen (hCol1) preparation has been reported to reduce pain in human OA and support a positive influence on chondrocyte function. To evaluate the tissue and cellular basis for these effects, we examined the impact of orally administered hCol1 in a model of posttraumatic OA (PTOA). In addition to standard chow, male C57BL/6J mice were provided a daily oral dietary supplement of hCol1 and a meniscal-ligamentous injury was induced on the right knee. At various time points post-injury, hydroxyproline (hProline) assays were performed on blood samples to confirm hCol1 delivery, and joints were harvested for tissue and molecular analyses were performed, including histomorphometry, OARSI and synovial scoring, immunohistochemistry and mRNA expression studies. Confirming ingestion of the supplements, serum hProline levels were elevated in experimental mice administered hCol1. In the hCol1 supplemented mice, chondroprotective effects were observed in injured knee joints, with dose-dependent increases in cartilage area, chondrocyte number and proteoglycan matrix at 3 and 12 weeks post-injury. Preservation of cartilage and increased chondrocyte numbers correlated with reductions in MMP13 protein levels and apoptosis, respectively. Supplemented mice also displayed reduced synovial hyperplasia that paralleled a reduction in Tnf mRNA, suggesting an anti-inflammatory effect. These findings establish that in the context of murine knee PTOA, daily oral consumption of hCol1 is chondroprotective, anti-apoptotic in articular chondrocytes, and anti-inflammatory. While the underlying mechanism driving these effects is yet to be determined, these findings provide the first tissue and cellular level information explaining the already published evidence of symptom relief supported by hCol1 in human knee OA. These results suggest that oral consumption of hCol1 is disease modifying in the context of PTOA.

Suppressive Effects of Insulin on Tumor Necrosis Factor-Dependent Early Osteoarthritic Changes Associated With Obesity and Type 2 Diabetes Mellitus.

OBJECTIVE: Obesity is a state of chronic inflammation that is associated with insulin resistance and type 2 diabetes mellitus (DM), as well as an increased risk of osteoarthritis (OA). This study was undertaken to define the links between obesity-associated inflammation, insulin resistance, and OA, by testing the hypotheses that 1) tumor necrosis factor (TNF) is critical in mediating these pathologic changes in OA, and 2) insulin has direct effects on the synovial joint that are compromised by insulin resistance. METHODS: The effects of TNF and insulin on catabolic gene expression were determined in fibroblast-like synoviocytes (FLS) isolated from human OA synovium. Synovial TNF expression and OA progression were examined in 2 mouse models, high-fat (HF) diet-fed obese mice with type 2 DM and TNF-knockout mice. Insulin resistance was investigated in synovium from patients with type 2 DM. RESULTS: Insulin receptors (IRs) were abundant in both mouse and human synovial membranes. Human OA FLS were insulin responsive, as indicated by the dose-dependent phosphorylation of IRs and Akt. In cultures of human OA FLS with exogenous TNF, the expression and release of MMP1, MMP13, and ADAMTS4 by FLS were markedly increased, whereas after treatment with insulin, these effects were selectively inhibited by >50%. The expression of TNF and its abundance in the synovium were elevated in samples from obese mice with type 2 DM. In TNF-knockout mice, increases in osteophyte formation and synovial hyperplasia associated with the HF diet were blunted. The synovium from OA patients with type 2 DM contained markedly more macrophages and showed elevated TNF levels as compared to the synovium from OA patients without diabetes. Moreover, insulin-dependent phosphorylation of IRs and Akt was blunted in cultures of OA FLS from patients with type 2 DM. CONCLUSION: TNF appears to be involved in mediating the advanced progression of OA seen in type 2 DM. While insulin plays a protective, antiinflammatory role in the synovium, insulin resistance in patients with type 2 DM may impair this protective effect and promote the progression of OA.