Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by aligners: a pilot study.

INTRODUCTION: The search for more aesthetic and comfortable orthodontic devices has led to an increase in the use of clear aligners. OBJECTIVE: To increase knowledge on biological mechanisms of orthodontic tooth movement using Invisalign aligners. METHODS: This study included 11 patients with a mean age of 23.6 ± 4.8 years. Cases planning included alignment and leveling of lower incisors using Invisalign aligners. Gingival crevicular fluid samples were collected from the lower incisors on the day of delivery of aligner number 1 (T0) and after 1 (T24h), 7 (T7d), and 21 (T21d) days. During the observation period of the study, the patients used only the aligner number 1. Levels of nine cytokines were quantified using Luminex's multi-analysis technology. Non-parametric tests were used for comparisons between cytokine expression levels over time. RESULTS: Cytokine expression levels remained constant after 21 days of orthodontic activation, except those of MIP-1ß, which presented a statistical difference between T24h and T21d with a decrease in the concentration levels. IL-8, GM-CSF, IL-1ß, MIP-1ß, and TNF-α showed the highest concentrations over time. CONCLUSIONS: The different behavior in the levels of the investigated cytokines indicates a role of these biomarkers in the tissue remodeling induced by Invisalign.

Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by aligners: a pilot study

ABSTRACT Introduction: The search for more aesthetic and comfortable orthodontic devices has led to an increase in the use of clear aligners. Objective: To increase knowledge on biological mechanisms of orthodontic tooth movement using Invisalign aligners. Methods: This study included 11 patients with a mean age of 23.6 ± 4.8 years. Cases planning included alignment and leveling of lower incisors using Invisalign aligners. Gingival crevicular fluid samples were collected from the lower incisors on the day of delivery of aligner number 1 (T0) and after 1 (T24h), 7 (T7d), and 21 (T21d) days. During the observation period of the study, the patients used only the aligner number 1. Levels of nine cytokines were quantified using Luminex's multi-analysis technology. Non-parametric tests were used for comparisons between cytokine expression levels over time. Results: Cytokine expression levels remained constant after 21 days of orthodontic activation, except those of MIP-1β, which presented a statistical difference between T24h and T21d with a decrease in the concentration levels. IL-8, GM-CSF, IL-1β, MIP-1β, and TNF-α showed the highest concentrations over time. Conclusions: The different behavior in the levels of the investigated cytokines indicates a role of these biomarkers in the tissue remodeling induced by Invisalign.

RESUMO Introdução: a busca por dispositivos ortodônticos mais estéticos e confortáveis gerou um aumento no uso de alinhadores transparentes. Objetivo: ampliar o conhecimento sobre os mecanismos biológicos associados ao movimento dentário ortodôntico promovido por alinhadores Invisalign®. Métodos: a amostra foi constituída por 11 pacientes, com idade média de 23,6 ± 4,8 anos. O planejamento dos casos incluiu alinhamento e nivelamento de incisivos inferiores usando os alinhadores. O fluido gengival crevicular foi coletado na superfície vestibular de incisivos inferiores no dia da entrega do alinhador número 1 (T0) e após 1 (T24h), 7 (T7d) e 21 (T21d) dias. Durante o período de observação do estudo, os pacientes utilizaram apenas o alinhador número 1. Os níveis de nove citocinas foram quantificados por meio do sistema Luminex de multianálise. Testes não paramétricos foram realizados para comparações entre os níveis de expressão de citocinas ao longo do tempo. Resultados: a concentração das citocinas manteve-se constante após 21 dias de ativação ortodôntica, exceto a MIP-1β, que apresentou uma redução estatisticamente significativa entre os tempos T24h e T21d. As IL-8, GM-CSF, IL-1β, MIP-1β e TNF-α apresentaram as maiores concentrações ao longo do tempo. Conclusão: a constância na expressão dos níveis das citocinas parece estar compatível com o estímulo mecânico induzido por alinhadores.

[Undifferentiated carcinoma of the jejunum producing granulocyte colony-stimulating factor].

An 80-year-old man presented with abdominal fullness and vomiting. Laboratory data revealed severe anemia, an inflammatory response, and elevated white blood cell counts. Abdominal computed tomography indicated ileus caused by a jejunal tumor measuring 8cm in diameter. Although small-bowel endoscopy enabled visualization of the tumor, adequate biopsy specimens could not be obtained for accurate diagnosis. The patient's condition rapidly deteriorated, because of which surgical treatment could not be initiated. The patient died approximately 3 weeks after admission. High serum granulocyte colony-stimulating factor (G-CSF) levels were detected at autopsy. Immunohistochemical staining of the autopsy specimen indicated positive G-CSF levels in the jejunal tumor. On the basis of these findings, a final diagnosis of undifferentiated carcinoma of the jejunum producing G-CSF was made.

A direct-infusion- and HPLC-ESI-Orbitrap-MS approach for the characterization of intact PEGylated proteins.

The characterization of proteins modified with poly(ethylene glycol) (PEG), such as recombinant human granulocyte-colony stimulating factor (PEGylated rhG-CSF or pegfilgrastim), by electrospray ionization-mass spectrometry (ESI-MS) constitutes a challenge due to the overlapping protein charge state pattern and PEG polydispersity. In order to minimize spectral overlaps, charge reduction by means of the addition of amine was applied. Method development for direct-infusion measurements, carried out on an ESI-time-of-flight (ESI-TOF) instrument, demonstrated the potential of triethylamine (TEA) for shifting the charge state pattern toward lower-charged species and of formic acid (FA) for causing higher charging. After successful method transfer to the LTQ Orbitrap XL instrument, isotopically resolved mass spectra could be acquired. With a median mass accuracy of 1.26 ppm, a number-average monoisotopic molecular mass of 40074.64 Da was determined for pegfilgrastim. The direct comparison of three Orbitrap mass spectrometers, namely the LTQ Orbitrap XL, the Exactive, and the Q Exactive, demonstrated that online interfacing to high performance liquid chromatography (HPLC) was only feasible with the Q Exactive, which offers adequate spectral quality on a time scale compatible with chromatographic separation (i.e., 0.2 min acquisition time per chromatographic peak). Finally, the applicability of both direct-infusion Orbitrap MS and HPLC interfaced to Orbitrap MS was demonstrated for the detection of methionine oxidation in pegfilgrastim. Singly, doubly, and triply oxidized species were readily resolved in the chromatogram, while their oxidation status was easily determined from the mass shifts observed in the deconvoluted mass spectra.

Low-dose radiation (LDR) induces hematopoietic hormesis: LDR-induced mobilization of hematopoietic progenitor cells into peripheral blood circulation.

OBJECTIVE: The aim of this study was to investigate the stimulating effect of low-dose radiation (LDR) on bone marrow hematopoietic progenitor cell (HPC) proliferation and peripheral blood mobilization. METHODS: Mice were exposed to 25- to 100-mGy x-rays. Bone marrow and peripheral blood HPCs (BFU-E, CFU-GM, and c-kit+ cells) were measured, and GM-CSF, G-CSF, and IL-3 protein and mRNA expression were detected using ELISA, slot blot hybridization, and Northern blot methods. To functionally evaluate LDR-stimulated and -mobilized HPCs, repopulation of peripheral blood cells in lethally irradiated recipients after transplantation of LDR-treated donor HPCs was examined by WBC counts, animal survival, and colony-forming units in the recipient spleens (CFUs-S). RESULTS: 75-mGy x-rays induced a maximal stimulation for bone marrow HPC proliferation (CFU-GM and BFU-E formation) 48 hours postirradiation, along with a significant increase in HPC mobilization into peripheral blood 48 to 72 hours postradiation, as shown by increases in CFU-GM formation and proportion of c-kit+ cells in the peripheral mononuclear cells. 75-mGy x-rays also maximally induced increases in G-CSF and GM-CSF mRNA expression in splenocytes and levels of serum GM-CSF. To define the critical role of these hematopoietic-stimulating factors in HPC peripheral mobilization, direct administration of G-CSF at a dose of 300 microg/kg/day or 150 microg/kg/day was applied and found to significantly stimulate GM-CFU formation and increase c-kit+ cells in the peripheral mononuclear cells. More importantly, 75-mGy x-rays plus 150 microg/kg/day G-CSF (LDR/150-G-CSF) produced a similar effect to that of 300 microg/kg/day G-CSF alone. Furthermore, the capability of LDR-mobilized donor HPCs to repopulate blood cells was confirmed in lethally irradiated recipient mice by counting peripheral WBC and CFUs-S. CONCLUSION: These results suggest that LDR induces hematopoietic hormesis, as demonstrated by HPC proliferation and peripheral mobilization, providing a potential approach to clinical application for HPC peripheral mobilization.

Persistent infection of human thymic epithelial cells by coxsackievirus B4.

Persistent replication of coxsackievirus B4 (CVB4) E2 (diabetogenic) and CVB4 JBV (nondiabetogenic) strains in thymic epithelial cell (TEC)-enriched cultures (>or=95%) was proved by detection of positive- and negative-strand viral RNA by reverse transcription-PCR in extracted RNA from cell cultures, VP1 capsid protein detection by immunofluorescence (IF) staining, and release of infectious particles up to 30 days after infection without obvious cytolysis. By double-IF staining, cytokeratin-containing cells were shown to be susceptible to CVB4. The persistence of CVB4 was associated with a significantly increased rate of TEC proliferation (up to 70%) after 20 days of culture and a significantly increased chronic production of immunoreactive interleukin-6 (IL-6), leukemia inhibitory factor, and granulocyte-macrophage colony-stimulating factor in supernatant after 3 days of culture. The CVB4 replication and the release of cytokines were not restricted to the CVB4 E2 diabetogenic strain and did not depend on the genetic background of the host; however, TEC were more responsive to CVB4 E2 than CVB4 JBV as far as the production of cytokines.

[Study of mechanisms of anti-irradiation effects of interleukin-1beta in long-term bone marrow cultures]. / Issledovanie mekhanizmov protivoluchevogo deistviia interleikina-1beta na modeli dlitel'nykh kul'tur kostnogo mozga.

The influence of betaleukin (human recombinant interleukin-1 beta) on the processes of postirradiation recovery of haemopoietic precursors (GM-CFC) and the level of granulocyte-macrophag colony-stimulating factor (GM-CSF) were studied in long-term bone marrow cultures after gamma-irradiation with a dose 2 Gy. Then the betaleukin action on the contents of GM-CFC and induction of GM-CSF in the non irradiated cultures was studied. It was shown that betaleukin increased the induction of GM-CSF and raised the contents of GM-CFC in long-term bone marrow cultures, and the maximal increase of a GM-CSF level and GM-CFC amount was marked in 20 hours after introduction. At an irradiation of long-term bone marrow cultures in conditions of betaleukin introduction 20 hours prior to influence of radiation the smaller degree of damage and faster recovery of GM-CFC was observed. The data in this report suggest that one of the mechanisms of antiirradiation action of betaleukin apparently is connected to the action of the preparation on hematopoietic microenvironment cellular elements, that causes the release of a colony-stimulating factor and stimulation of recovery of haemopoietic precursors.

Cytokines, chemokines, and colony-stimulating factors in human milk: the 1997 update.

Epidemiologic studies conducted over the past 30 years to investigate the protective functions of human milk strongly support the notion that breast-feeding prevents infantile infections, particularly those affecting the gastrointestinal and respiratory tracts. However, more recent clinical and experimental observations also suggest that human milk not only provides passive protection, but also can directly modulate the immunological development of the recipient infant. The study of this remarkable defense system in human milk has been difficult due to its biochemical complexity, the small concentration of certain bioactive components, the compartmentalization of some of these agents, the dynamic quantitative and qualitative changes of milk during lactation, and the lack of specific reagents to quantify these agents. Nevertheless, a host of bioactive substances including hormones, growth factors, and immunological factors such as cytokines have been identified in human milk. Cytokines are pluripotent polypeptides that act in autocrine/paracrine fashions by binding to specific cellular receptors. They operate in networks and orchestrate the development and functions of the immune system. Several different cytokines and chemokines have been discovered in human milk over the past years, and the list is growing very rapidly. This article will review the current knowledge about the increasingly complex network of chemoattractants, activators, and anti-inflammatory cytokines present in human milk and their potential role in compensating for the developmental delay of the neonate immune system.

Modulation of hemopoietic factor production in relation to endothelial cell aging by interleukin-1 induction.

In this study, we examined the modulation of hemopoietic factor production by human umbilical vein endothelial cells in relation to aging and the cell cycle under conditions of interleukin-1 (IL-1) induction and noninduction. Under conditions of IL-1 noninduction, messenger RNA expression levels of macrophage colony-stimulating factor (M-CSF) were three times higher in non-S-phase cells of young cultures than those in S-phase cells. Expression levels decreased in non-S-phase cells of old culture and approached levels similar to that of S-phase cells. The expression of neither E-selectin nor erythropoietin (Epo) was detected in cells from the noninduced state. The expression of granulocyte colony-stimulating factor (G-CSF) was not affected by either cellular aging or the cell cycle; however, the amount of product secreted increased significantly in old cells, suggesting that G-CSF production is under posttranscriptional regulation. Under conditions of IL-1 induction G-CSF and M-CSF expression levels were enhanced in both young and old cells. Expression of Epo was not detected whereas E-selectin was induced. Significant M-CSF product was detected in young cells but not in old cells, whereas G-CSF product increased dramatically in both types of cells. The modulation of these factors is discussed in relation to the maintenance of neutrophil concentration, differentiation, and maturation of leukocytes and their possible effect on atherosclerosis.

Purification and characterization of thrombopoietin.

A thrombopoietic factor, termed thrombopoietin (TPO), was highly purified directly from the plasma of sublethally irradiated 1,100 rats by measuring the production of megakaryocytes from a highly enriched population of rat megakaryocyte progenitor cells (CFU-MK). The rat plasma TPO is a glycoprotein and strongly hydrophobic. The total activity and purification yields obtained were about 29% and 1.49 x 10(8), respectively. The amino acid sequences of the two peptide fragments prepared from the purified 19 kDa TPO were analyzed, and used for the cloning of rat and human TPO cDNAs. It was found that the 19 kDa TPO was truncated but comprised at least 163 amino acids. The sequence of human TPO cDNA revealed that the TPO was identical to the c-Mpl ligand. Both rat and human TPOs expressed in COS-1 cells exhibited significant activity toward the CFU-MK in vitro, and were active in stimulating platelet production in mice. These results indicate that a thrombopoietic factor originally found in the irradiated rat plasma is a ligand for the rat c-Mpl.

The detection of colony-stimulating factors and steel factor in adherent layers of human long-term marrow cultures using reverse-transcriptase polymerase chain reaction.

In human long-term marrow cultures granulomonopoiesis is maintained for several weeks. Studies on granulomonocytic progenitors (CFU-GM) and their progeny have shown that survival, proliferation, differentiation and maturation of these cells are controlled by a set of glycoproteins, the colony-stimulating factors (CSFs) and the Steel factor. We have studied the expression of these factors using reverse transcriptase polymerase chain reaction (RT-PCR) in 17 adherent layers of normal bone marrow at 3, 5 or 7 weeks of culture. We have taken the 5637 bladder carcinoma cell line as a control for expression of GM-CSF, M-CSF, G-CSF and Steel factor, and PHA-activated T lymphocytes as a control for expression of multi-CSF (interleukin 3, IL-3). We have found that GM-CSF was expressed in the 17 adherent layers without induction by interleukin 1 beta (IL-1 beta). M-CSF was also detected in all cases, but in two early-stage (week 3 and week 5) cultures only after stimulation by IL-1 beta. G-CSF was detected in only 11 cases (three without IL-1 beta, and eight after addition of IL-1 beta). Steel factor was detected in 14 cases (ten without IL-1 beta, and four after addition of IL-1 beta). IL-3 was not detected even by means of nested RT-PCR. These data indicate in six late-stage (week 5 or week 7) cultures G-CSF messenger concentrations 10(3)-fold less than in 5637 control cells (for an identical amount of total cellular RNA). A similar conclusion may be drawn for Steel factor in three late-stage cultures. For IL-3 our negative results indicate a messenger concentration 10(5)-fold less than in activated T lymphocytes. These results suggest a crucial role for GM-CSF and M-CSF in the maintenance of granulomonopoiesis in human long-term cultures. The role of G-CSF and Steel factor may be more marginal. Eventually IL-2 may not be involved in the regulatory process.

Relative roles of osteoclast colony-stimulating factor and macrophage colony-stimulating factor in the course of osteoclast development.

Although recent studies have shown that osteopetrotic (op/op) mice lack macrophage colony-stimulating factor (M-CSF or CSF-1), the precise role of M-CSF in the development of immature osteoclasts remains unknown. Using a recently discovered osteoclast-specific colony-stimulating factor (O-CSF) and in vitro long-term bone marrow culture systems, we investigated the ability of op/op and control marrow stromal cells to support the production of O-CSF-responsive clonogenic osteoclast progenitors (colony-forming unit-osteoclast [CFU-O]) from inoculated normal stem cells. Remarkably, op/op stromal cell cultures produced five times as many nonadherent cells as control cultures throughout the experimental period of 14 weeks; an average of 37% of these cells were nonviable compared with 8% in control cultures. Significantly higher numbers of CFU-O were found in op/op cultures than in control cultures; the CFU-O in op/op and control cultures were proliferating at a similar rate. Higher numbers of calcitonin receptor-bearing cells were found when harvested cells from op/op flasks were cultured with 1,25(OH)2D3. These studies clearly show that op/op marrow stromal cells can support the differentiation and proliferation of osteoclast progenitors from inoculated stem cells and provide the first experimental evidence that M-CSF is not essential for the early stages of osteoclast development. We hypothesize that while O-CSF supports proliferation of osteoclast progenitors, M-CSF plays a role in the later development and maturation of the progenitor as well as in the prevention of cell death.

A sensitive new bioassay for erythroid colony-stimulating factor.

Erythroid colony-stimulating factor (E-CSF) is a B cell-derived membrane protein that specifically affects the growth and development of human and murine committed erythroid progenitors. We report the development of a sensitive new bioassay for E-CSF, based on the ability of the growth factor to stimulate 3H-thymidine incorporation into cloned Rauscher murine erythroleukemia cells. The assay has among its advantages the ability to measure growth factor activity on a purified target cell population in the absence of endogenous growth factor-producing accessory cells. In addition, this assay measures E-CSF's proliferative effect on erythroid progenitors in the absence of erythropoietin (Epo) after 72 to 96 hours. In contrast, the standard bone marrow fibrin clot assay traditionally used to measure E-CSF requires the addition of Epo to promote the development of hemoglobinized erythroid colonies that are quantified after 7 days (for murine cells) to 12 days (for human cells). With the use of this new Rauscher cell bioassay, we have identified an E-CSF-producing human cell line and, further, have measured E-CSF activity derived from nonhuman splenic B lymphocytes.

B-lymphocyte-derived burst-promoting activity is a pleiotropic erythroid colony-stimulating factor, E-CSF.

Human B-lymphocyte-derived erythroid burst-promoting activity (B-BPA) is a pleiotropic, lineage-specific regulator of erythropoiesis. Our present data indicate that B-BPA plays an important role as an erythroid colony-stimulating factor (E-CSF) in modulating progenitor growth and differentiation throughout erythropoiesis. E-CSF has discrete effects on both early (erythroid burst-forming units, BFU-E) and late (erythroid colony-forming units, CFU-E) progenitors from normal bone marrow. In serum-substituted fibrin clot cultures, E-CSF stimulates the proliferation of BFU-E, resulting in an increase in the number of erythroid bursts over a wide range of erythropoietin (Epo) concentrations. We now have shown that E-CSF also acts on CFU-E by increasing their sensitivity to Epo markedly, resulting in a tenfold left-shift in the Epo dose-response curve. Using purified target-cell populations of human and murine erythroleukemia cells that are Epo-independent for growth, we have found that E-CSF stimulates cell proliferation directly, increasing the plating efficiency of these cells in suspension culture by 50%-165%. B-BPA also increased proliferation of these cells in semi-solid medium. Importantly, the combination of E-CSF and Epo resulted in a profound increase in the growth and maturation of the resultant colonies. Therefore, the data indicate that E-CSF can regulate the growth of cells independently of added Epo and, in addition, can synergize with Epo in regulating the growth and differentiation of erythroid progenitors.

Thymic hyperplasia in transgenic mice caused by immortal epithelial cells expressing c-kit ligand.

To dissect mechanisms that co-ordinate specific events in thymopoiesis we have characterized alterations in thymic structure and function caused by expression of a transgene. This gene encodes SV40Tag and is specifically expressed in a subset of thymic epithelial (TE) cells around birth. As a result the number of immortal TE cells increases, thymic mass increases (up to 3 g), and thymopoiesis is expanded. The latter is reflected by a approximately 100-fold increase of the major thymocyte subsets and increased peripheral T cell counts. Grossly hyperplastic thymi retain many but not all morphological features of a normal thymus. Also in grafts, SV40Tag+ TE cells steer expansion (up to 8 g) and organize a tissue with mainly cortex-like features that includes mainly SV40Tag+ TE cells, thymocytes, and macrophages. To investigate expression of specialized gene functions in the immortal TE cells, a cell line was derived. The Epi-A1 cell line expresses the genes for major histocompatibility complex class I and II, Thy-1, interleukin (IL)-6, IL-7, macrophage-colony-stimulating factor, and transforming growth factor-beta 3. Most importantly, Epi-A1 cells also express the IL-4 receptor and the c-kit ligand (KL), a factor that, in concert with commitment factors, channels progenitors into hemopoietic lineages. The expression of low constitutive levels of KL mRNA does not require IL-4, but KL mRNA levels are increased dramatically in response to IL-4. Since constitutive expression of KL mRNA in vivo is restricted to a small subset of TE cells in the thymus, our findings reveal a novel specific interaction between thymocytes and a specialized subset of TE cells.