Performance of Multiplex Cytokine Assays in Serum and Saliva among Community-Dwelling Postmenopausal Women

Multiplexing arrays increase the throughput and decrease sample requirements for studies employing multiple biomarkers. The goal of this project was to examine the performance of Multiplex arrays for measuring multiple protein biomarkers in saliva and serum. Specimens from the OsteoPerio ancillary study of the Women’s Health Initiative Observational Study were used. Participants required the presence of at least 6 teeth and were excluded based on active cancer and certain bone issues but were not selected on any specific condition. Quality control (QC) samples were created from pooled serum and saliva. Twenty protein markers were measured on five multiplexing array panels. Sample pretreatment conditions were optimized for each panel. Recovery, lower limit of quantification (LLOQ) and imprecision were determined for each analyte. Statistical adjustment at the plate level was used to reduce imprecision estimates and increase the number of usable observations. Sample pre-treatment improved recovery estimates for many analytes. The LLOQ for each analyte agreed with manufacturer specifications except for MMP-1 and MMP-2 which were significantly higher than reported. Following batch adjustment, 17 of 20 biomarkers in serum and 9 of 20 biomarkers in saliva demonstrated acceptable precision, defined as <20% coefficient of variation (<25% at LLOQ). The percentage of cohort samples having levels within the reportable range for each analyte varied from 10% to 100%. The ratio of levels in saliva to serum varied from 1∶100 to 28∶1. Correlations between saliva and serum were of moderate positive magnitude and significant for CRP, MMP-2, insulin, adiponectin, GM-CSF and IL-5. Multiplex arrays exhibit high levels of analytical imprecision, particularly at the batch level. Careful sample pre-treatment can enhance recovery and reduce imprecision. Following statistical adjustments to reduce batch effects, we identified biomarkers that are of acceptable quality in serum and to a lesser degree in saliva using Multiplex arrays.     

Localized aggressive periodontitis is linked to human chromosome 1q25

Localized aggressive periodontitis (LAP; previously known as localized juvenile periodontitis) is one of the rapidly progressive periodontal diseases. Certain forms of familial LAP show a simple Mendelian pattern of transmission. However, no gene mutation has been identified to be responsible for the LAP phenotype. As an initial step to identify a gene mutation associated with LAP, we have performed genetic linkage analysis with four multigenerational families exhibiting the LAP phenotype. Affected individuals in the families were identified based on clinical and laboratory criteria in an attempt to define a homogeneous phenotype, since the clinical presentation of LAP may represent a manifestation of a heterogeneous group of diseases. The LAP phenotype is linked to a DNA marker, D1S492, with LOD score 3.48, =0.00. The haplotype analysis of the chromosome interval associated with D1S492 indicates that a LAP locus is located between D1S196 and D1S533 on chromosome 1, covering about 26 million DNA basepairs. We have also examined the DNA sequence of prostaglandin-endoperoxide synthase 2 (PTGS2 or cyclooxygenase 2, COX2) since prostaglandin 2 (PGE2), the product of COX2, is upregulated in LAP patients and COX2 is located between D1S196 and D1S533. No mutation in COX2 was identified in the patients.     

Immune response of macrophages from young and aged mice to the oral pathogenic bacterium <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>

Periodontal disease is a chronic inflammatory gum disease that in severe cases leads to tooth loss. Porphyromonas gingivalis (Pg) is a bacterium closely associated with generalized forms of periodontal disease. Clinical onset of generalized periodontal disease commonly presents in individuals over the age of 40. Little is known regarding the effect of aging on inflammation associated with periodontal disease. In the present study we examined the immune response of bone marrow derived macrophages (BMM) from young (2-months) and aged (1-year and 2-years) mice to Pg strain 381. Pg induced robust expression of cytokines; tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10, chemokines; neutrophil chemoattractant protein (KC), macrophage colony stimulating factor (MCP)-1, macrophage inflammatory protein (MIP)-1α and regulated upon activation normal T cell expressed and secreted (RANTES), as well as nitric oxide (NO, measured as nitrite), and prostaglandin E2 (PGE2) from BMM of young mice. BMM from the 2-year age group produced significantly less TNF-α, IL-6 and NO in response to Pg as compared with BMM from 2-months and 1-year of age. We did not observe any difference in the levels of IL-1β, IL-10 and PGE2 produced by BMM in response to Pg. BMM from 2-months and 1-year of age produced similar levels of all chemokines measured with the exception of MCP-1, which was reduced in BMM from 1-year of age. BMM from the 2-year group produced significantly less MCP-1 and MIP-1α compared with 2-months and 1-year age groups. No difference in RANTES production was observed between age groups. Employing a Pg attenuated mutant, deficient in major fimbriae (Pg DPG3), we observed reduced ability of the mutant to stimulate inflammatory mediator expression from BMMs as compared to Pg 381, irrespective of age. Taken together these results support senescence as an important facet of the reduced immunological response observed by BMM of aged host to the periodontal pathogen Pg.     

Chronic hyperglycemia predisposes to exaggerated inflammatory response and leukocyte dysfunction in Akita mice

The role of polymorphonuclear neutrophils (PMN) in mediating diabetic tissue damage to the periodontium was investigated in a novel model of chronic hyperglycemia, the Akita mouse. Induction of acute peritoneal inflammation in wild-type (WT) and Akita mice resulted in exaggerated IL-6 response in Akita mice (2.9-fold increase over WT values) and a markedly increased chemokine response (KC, 2.6-fold; MCP-1, 2.6-fold; and MIP-1alpha, 4.4-fold increase over WT values). Chemotaxis to both fMLP and WKYMVm was significantly reduced in isolated Akita PMN compared with WT PMN as measured in a Boyden chamber. Superoxide release in contrast was significantly increased in Akita PMN as measured with cytochrome c reduction. Bone marrow-derived Akita PMN showed partial translocation of p47phox to the cell membrane without external stimulation, suggesting premature assembly of the superoxide-producing NADPH oxidase in hyperglycemia. In vivo studies revealed that ligature-induced periodontal bone loss is significantly greater in Akita mice compared with WT. Moreover, intravital microscopy of gingival vessels showed that leukocyte rolling and attachment to the vascular endothelium is enhanced in periodontal vessels of Akita mice. These results indicate that chronic hyperglycemia predisposes to exaggerated inflammatory response and primes leukocytes for marginalization and superoxide production but not for transmigration. Thus, leukocyte defects in hyperglycemia may contribute to periodontal tissue damage by impairing the innate immune response to periodontal pathogens as well as by increasing free radical load in the gingival microvasculature.     

Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) affects in particular the T-cell lineage and is associated with an increased risk for infections, graft failure and malignant relapse. To generate large numbers of T-cell precursors for adoptive therapy, we cultured mouse hematopoietic stem cells (HSCs) in vitro on OP9 mouse stromal cells expressing the Notch-1 ligand Delta-like-1 (OP9-DL1). We infused these cells, together with T-cell-depleted mouse bone marrow or purified HSCs, into lethally irradiated allogeneic recipients and determined their effect on T-cell reconstitution after transplantation. Recipients of OP9-DL1-derived T-cell precursors showed increased thymic cellularity and substantially improved donor T-cell chimerism (versus recipients of bone marrow or HSCs only). OP9-DL1-derived T-cell precursors gave rise to host-tolerant CD4+ and CD8+ populations with normal T-cell antigen receptor repertoires, cytokine secretion and proliferative responses to antigen. Administration of OP9-DL1-derived T-cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity but not graft-versus-host disease (GVHD). We conclude that the adoptive transfer of OP9-DL1-derived T-cell precursors markedly enhances T-cell reconstitution after transplantation, resulting in GVT activity without GVHD.     

Reduced inflammation and tissue damage in transgenic rabbits overexpressing 15-lipoxygenase and endogenous anti-inflammatory lipid mediators

PGs and leukotrienes (LTs) mediate cardinal signs of inflammation; hence, their enzymes are targets of current anti-inflammatory therapies. Products of arachidonate 15-lipoxygenases (LO) types I and II display both beneficial roles, such as lipoxins (LXs) that stereoselectively signal counterregulation, as well as potential deleterious actions (i.e., nonspecific phospholipid degradation). In this study, we examined transgenic (TG) rabbits overexpressing 15-LO type I and their response to inflammatory challenge. Skin challenges with either LTB(4) or IL-8 showed that 15-LO TG rabbits give markedly reduced neutrophil (PMN) recruitment and plasma leakage at dermal sites with LTB(4). PMN from TG rabbits also exhibited a dramatic reduction in LTB(4)-stimulated granular mobilization that was not evident with peptide chemoattractants. Leukocytes from 15-LO TG rabbits gave enhanced LX production, underscoring differences in lipid mediator profiles compared with non-TG rabbits. Microbe-associated inflammation and leukocyte-mediated bone destruction were assessed by initiating acute periodontitis. 15-LO TG rabbits exhibited markedly reduced bone loss and local inflammation. Because enhanced LX production was associated with an increased anti-inflammatory status of 15-LO TG rabbits, a stable analog of 5S,6R,15S-trihydroxyeicosa-7E,9E,11Z,13E-tetraenoic acid (LXA(4)) was applied to the gingival crevice subject to periodontitis. Topical application with the 15-epi-16-phenoxy-para-fluoro-LXA(4) stable analog (ATLa) dramatically reduced leukocyte infiltration, ensuing bone loss as well as inflammation. These results indicate that overexpression of 15-LO type I and LXA(4) is associated with dampened PMN-mediated tissue degradation and bone loss, suggesting that enhanced anti-inflammation status is an active process. Moreover, they suggest that LXs can be targets for novel approaches to diseases, e.g., periodontitis and arthritis, where inflammation and bone destruction are features.     

IFN-gamma and Fas ligand are required for graft-versus-tumor activity against renal cell carcinoma in the absence of lethal graft-versus-host disease

To determine the mechanisms of graft-versus-tumor (GVT) activity in the absence of graft-versus-host disease (GVHD) against a solid tumor, we established two allogeneic bone marrow transplantation models with a murine renal cell carcinoma (RENCA). The addition of 0.3 x 10(6) donor CD8(+) T cells to the allograft increased the survival of tumor-bearing mice without causing GVHD. The analysis of CD8(+) T cells deficient in cytotoxic molecules demonstrated that anti-RENCA activity is dependent on IFN-gamma and Fas ligand (FasL), but does not require soluble or membrane-bound TNF-alpha, perforin, or TRAIL. Recipients of IFN-gamma(-/-) CD8(+) T cells are unable to reject RENCA compared with recipients of wild-type CD8(+) T cells and, importantly, neither group develops severe GVHD. IFN-gamma(-/-) CD8(+) T cells derived from transplanted mice are less able to kill RENCA cells in vitro, while pretreatment of RENCA cells with IFN-gamma enhances class I and FasL expression and rescues the lytic capacity of IFN-gamma(-/-) CD8(+) T cells. These results demonstrate that the addition of low numbers of selected donor CD8(+) T cells to the allograft can mediate GVT activity without lethal GVHD against murine renal cell carcinoma, and this GVT activity is dependent on IFN-gamma and FasL.     

Enhanced immune reconstitution by sex steroid ablation following allogeneic hemopoietic stem cell transplantation

Delayed immune reconstitution in adult recipients of allogeneic hemopoietic stem cell transplantations (HSCT) is related to age-induced thymic atrophy. Overcoming this paucity of T cell function is a major goal of clinical research but in the context of allogeneic transplants, any strategy must not exacerbate graft-vs-host disease (GVHD) yet ideally retain graft-vs-tumor (GVT) effects. We have shown sex steroid ablation reverses thymic atrophy and enhances T cell recovery in aged animals and in congenic bone marrow (BM) transplant but the latter does not have the complications of allogeneic T cell reactivity. We have examined whether sex steroid ablation promoted hemopoietic and T cell recovery following allogeneic HSCT and whether this benefit was negated by enhanced GVHD. BM and thymic cell numbers were significantly increased at 14 and 28 days after HSCT in castrated mice compared with sham-castrated controls. In the thymus, the numbers of donor-derived thymocytes and dendritic cells were significantly increased after HSCT and castration; donor-derived BM precursors and developing B cells were also significantly increased. Importantly, despite restoring T cell function, sex steroid inhibition did not exacerbate the development of GVHD or ameliorate GVT activity. Finally, IL-7 treatment in combination with castration had an additive effect on thymic cellularity following HSCT. These results indicate that sex steroid ablation can profoundly enhance thymic and hemopoietic recovery following allogeneic HSCT without increasing GVHD and maintaining GVT.