Evaluation of Clinical Parameters in Implant Maintenance Phase for Prevention of Peri-implantitis.

AIMS AND OBJECTIVES: Peri-implant mucositis and peri-implantitis are one of the common biological complications affecting implant success. The present study aimed to evaluate various clinical parameters during implant maintenance phase. MATERIALS AND METHODS: The study included patients undergoing implant maintenance phase for 1-year follow-up. The study consists of a total of forty individuals with age ranging from 35 to 65 years. They were further categorized into two subgroups on the basis of their history, i.e., Group 1: patients with no history of periodontitis before implant placement and Group 2: patients with a history of periodontitis before implant placement. Among the selected patients, a total of 98 implants were studied. All were individually evaluated for clinical parameters such as gingival index, pocket probing depth (PPD), and bleeding on probing (BOP). All the data obtained were tabulated and analyzed using statistical software SPSS version 18.0 for Windows (SPSS Inc., Chicago, USA). Quantitative analysis was done using t-test and Mann-Whitney U-test. RESULTS: The mean age of the patients in Group 1 and Group 2 was 58.6 and 62.8 years, respectively, with not much gender difference. The mean plaque index for Group 1 was 0.17 ± 0.20, while for Group 2, it was 0.24 ± 0.14. The mean PPD and mean BOP for Group 1 came to be 2.60 ± 0.42 and 0.42 ± 0.15, respectively, whereas for Group 2, it was 4.08 ± 0.30 and 0.39 ± 0.48, respectively. Only PPD was found to be statistically significantly different between both the groups. Group 1 showed 2.0% peri-implantitis, whereas Group 2 showed 28% peri-implantitis. CONCLUSION: Due to increased prevalence of peri-implantitis cases with the increase in usage of implants, it becomes imperative to look up to the etiological factors and contributing factors so that the incidence of these can be minimized.

Pterygoid Implant for Atrophic Posterior Maxilla.

Replacement of missing teeth in the posterior maxilla is always a challenge for the treating implant surgeon as the posterior maxilla has several obstacles in the form of quality, quantity, the anatomy of the maxillary sinus, and inaccessibility. To overcome these deficiencies, several surgical procedures such as sinus lift, bone augmentation, tilted implants, short implants, and zygomatic implants were tried. Since these procedures have their own limitations, pterygomaxillary region provides us an excellent place for placement of implant and rehabilitation of posterior maxilla. This case report describes the usage of the pterygomaxillary region for placement of the implant to restore atrophic posterior maxilla, without any additional surgical procedures.

Expression of the placental cytokines tumor necrosis factor alpha, interleukin 1beta, and interleukin 10 is increased in preeclampsia.

OBJECTIVE: We sought to determine whether placental cytokine expression is altered in patients with preeclampsia. STUDY DESIGN: Whole placental tissue was collected at cesarean delivery, and total ribonucleic acid was extracted. Reverse transcriptase-polymerase chain reaction was performed to determine cytokine expression. Product bands were quantitated by scanning densitometry, and results were expressed as a ratio of cytokine/housekeeping gene (cytokine expression index). Statistical analysis was performed by the Student t test and the Mann-Whitney U test. RESULTS: Placentas from 6 patients with preeclampsia and 4 normotensive patients were analyzed. Placental expression of interleukin 1beta and interleukin 10 was greater in preeclamptic women than in normotensive subjects (median interleukin 1beta cytokine expression index, 0.675; range, 0.394-0. 953; vs 0.106; range, 0.084-0.166; P =.011; median interleukin 10 cytokine expression index, 1.042; range, 0.672-1.192; vs 0.126; range, 0.062-0.398; P <.011). Tumor necrosis factor alpha messenger ribonucleic acid was detected in placentas of preeclamptic subjects but not in normotensive control subjects. CONCLUSION: Placentas from preeclamptic patients demonstrated increased expression of interleukin 1beta, interleukin 10, and tumor necrosis factor alpha. This may be in association with placental hypoxia and may contribute to the global endothelial dysfunction observed in preeclampsia.

First-trimester human chorionic villi express both immunoregulatory and inflammatory cytokines: a role for interleukin-10 in regulating the cytokine network of pregnancy.

PROBLEM: T-helper 2 (TH2)-type cytokines [i.e., interleukin (IL)-6, IL-10, and IL-13] and transforming growth factor (TGF)-beta are expressed by the murine decidua and/or placenta and are likely to suppress inflammatory cytokine [i.e., IL-2, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, IL-1 alpha, and IL-1 beta] production at the maternal-fetal interface. In addition, class I IFNs may protect the fetus from immunologic rejection and viral infections. This study examines the expression of inflammatory/immunoregulatory cytokines and IL-10 production by first-trimester chorionic villi. METHOD OF STUDY: Gestational tissues (n = 5) were obtained following elective terminations performed between 7 and 9 weeks of gestation. Chorionic villous tissues were separated from fetal membranes and decidua, and total RNA was extracted. Cytokine expression was assessed by a reverse transcriptase-polymerase chain reaction technique. Chorionic villi (n = 9; 6-12 weeks gestation) were maintained in organ culture, and human chorionic gonadotropin (hCG) and IL-10 levels were determined by immunoradiometric and enzyme-linked immunosorbent assays, respectively. RESULTS: IFN-gamma and IL-2 were generally not expressed by first-trimester chorionic villi. Low to moderate levels of expression were noted for IL-1 alpha, IL-1 beta, and TNF-alpha. High levels of mRNA were noted for IFN-alpha and IFN-beta, but IFN-tau was not expressed. In all tissues, TGF-beta 1 and IL-13 were either weakly expressed or not expressed. In contrast, moderate to high levels of IL-6 and IL-10 mRNA were detected in each chorionic villous sample. In chorionic villous explants obtained at 6-11 weeks gestation production of hCG and IL-10 was greatest during the first 24 hr ([hCG] = 6961 +/- 815 mIU/mL, [IL-10] = 92 +/- 11 pg/mL) and then declined through 72 hr. CONCLUSIONS: TH1-type cytokines (IL-2, IFN-gamma) are not expressed by first-trimester chorionic villous tissues: This is possibly due to local production of IL-10. In contrast, macrophage-associated cytokines (IL-1 beta and TNF-alpha) are expressed and their regulation may be critical for fetal survival. Finally, class 1 IFNs expressed by early chorionic tissues may protect the fetus from maternal rejection and viral transmission.

Cytokine expression by first-trimester human chorionic villi.

PROBLEM: Communication at the human maternal-fetal interface occurs by an intricate cytokine network. This study examines cytokine expression by normal first-trimester human chorionic villi. METHOD OF STUDY: Tissues were obtained at elective pregnancy terminations (7-9 weeks). Total RNA was isolated from chorionic villi by guanidinium isothiocynate-acid phenol extraction. A reverse transcriptase-polymerase chain reaction technique was used to examine cytokine expression. beta-Actin was used as the housekeeping gene, and mitogen-stimulated lymphocytes served as positive controls. RESULTS: beta-Actin was uniformly expressed by all chorionic villous samples. Interferon (IFN)-alpha and -beta also were highly expressed. Moderate expression was noted for interleukin (IL)-10, IL-6, tumor necrosis factor (TNF)-alpha, and IL-1 beta. In contrast, transforming growth factor-beta 1, IFN-gamma, IL-2, and IL-1 alpha were either weakly expressed or absent in first-trimester villi. CONCLUSIONS: Cytokines may contribute to pregnancy immunotolerance (IFN-alpha, IFN-beta, and IL-10), viral resistance (IFNs), hormone secretion (IL-1 and IL-6), and cellular remodeling (IFN-gamma and TNF-alpha) within the chorionic villous.

Expression and production of interleukin-10 by human trophoblast: relationship to pregnancy immunotolerance.

Interleukin-10 (IL-10) is a T-helper type-2 (Th2) cytokine noted for its ability to suppress cytokine synthesis by T-helper type-1 (Th1) cells. IL-10 may play a role in pregnancy immunotolerance through the establishment of a Th2 cytokine bias at the maternal-fetal interface. This study examines the expression and production of IL-10 by normal and malignant human trophoblast. Term placental biopsies, cloned choriocarcinoma cell lines and isolated human trophoblast were utilized for the study of IL-10 expression. Choriocarcinoma cells (BeWo, JEG-3, JAR) were maintained in T-flask culture until confluence and then harvested by enzymatic dispersion. Purified term trophoblast were obtained by sequential trypsin/DNAse digests and CD9 immunoaffinity chromatography. Amplified IL-10 mRNA was detected by a reverse transcriptase polymerase chain reaction (RTPCR) technique. BeWo cells were maintained in artificial capillary culture (ACC) and conditioned media assayed for IL-10. Granulocyte macrophage-colony stimulating factor (GM-CSF; 1.0, 10.0 and 100.0 ng/ml) was added to the BeWo cultures to examine its effects on trophoblast IL-10 production. IL-10 determinations were performed using a human ELISA system. IL-10 mRNA was detected in each trophoblast cell type examined with the exception of the JEG-3 choriocarcinoma cell line. IL-10 protein was also detected (range 6-22 pg/ml) in BeWo media on days 8 to 11 of culture. When serum was reduced in the culture media, IL-10 levels fell below the sensitivity of the assay (5 pg/ml). Subsequent addition of GM-CSF stimulated BeWo IL-10 secretion in a dose-related fashion. These results support the concept IL-10 is expressed at the human maternal-fetal interface, and production of this important immunoregulatory molecule may be regulated, in part, by GM-CSF.

In vivo molecular analysis of cytokines in a murine model of ocular onchocerciasis. I. Up-regulation of IL-4 and IL-5 mRNAs and not IL-2 and IFN gamma mRNAs in the cornea due to experimental interstitial keratitis.

Sclerosing keratitis is the major cause of blindness due to onchocerciasis which results from chronic infection with the filarial parasite Onchocerca volvulus. Using a murine model of onchocercal sclerosing keratitis, we have demonstrated previously that predominantly (> 85%) CD3 + /CD4+ T-cells as well as the IL-2 receptor bearing cells infiltrate into the cornea in vivo during development and progress of the disease. The identification of CD4+ subsets TH1 and TH2 based on the cytokine secretion patterns of murine T-lymphocytes has been useful for understanding the immune basis of resistance and pathogenesis in murine models of several parasitic diseases. The present investigation was carried out to demonstrate whether the local immune response at the corneal lesion due to onchocercal interstitial keratitis correlated with such distinct patterns of cytokine production. For that purpose, mRNA was extracted separately from corneas obtained from the diseased eyes and the normal eyes of A/J mice with onchocercal interstitial keratitis, reverse transcribed and amplified by the polymerase chain reaction with four different cytokine specific primers. In corneas obtained from the eyes affected with onchocercal interstitial keratitis, mRNAs coding for IL-4 and IL-5 were up-regulated compared to the normal eyes having no lesions from the same animals. However, the levels of mRNAs for IL-2 and IFN gamma were found to be the same in the diseased and normal eyes. Taken together, these data suggest that IL-4 and IL-5 producing TH2-lymphocytes are active at the corneal lesion due to onchocercal interstitial keratitis.

Cytokine expression by models of human trophoblast as assessed by a semiquantitative reverse transcription-polymerase chain reaction technique.

PROBLEM: Cytokines form an important communication network between the mother and fetus. Defining the significance of these factors requires an understanding of their constitutive expression by maternal and fetal tissues. This study examines cytokine expression by human trophoblast. METHODS: A reverse transcription polymerase chain reaction (RT-PCR) technique was used to assess cytokine expression by choriocarcinoma cells (BeWo, JEG-3, and JAR) and term trophoblast. Placental digests were enriched for trophoblast by immunoaffinity (CD-9) columns. RESULTS: Interleukin (IL)-1, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were weakly expressed or absent in the choriocarcinoma cells. In contrast, these cytokines were expressed by term trophoblast. IL-6, IL-10, IFN-alpha, IFN-beta, and granulocyte macrophage-colony stimulating factor (GM-CSF) mRNA were detected in all trophoblast cells, except for a paucity of IL-10 expression by JEG-3 cells. CONCLUSIONS: Human choriocarcinoma cells and term trophoblast express cytokines that may regulate critical reproductive events. Expression of inflammatory cytokines such as IL-1, TNF-alpha, and IFN-gamma by term trophoblast could trigger labor or be a consequence of labor-associated events.

Semiquantitative measurement of cytokine messenger RNA in endomyocardium and peripheral blood mononuclear cells from human heart transplant recipients.

BACKGROUND: Cytokines play a central role in inflammatory responses and in specific immune responses directed toward alloantigens. The pattern and quantity of cytokines produced in graft rejection can yield valuable information regarding the cellular and molecular mechanisms of the antigraft response. METHODS: We used the polymerase chain reaction to semiquantitatively measure changes in the amount of messenger RNA from the interleukin-1 beta, interleukin-2, interleukin-4, interleukin-6, interleukin-10, tumor necrosis factor-alpha, interferon-gamma, interleukin-1 receptor antagonist, and the interleukin-2 receptor genes in the peripheral blood and endomyocardium of cardiac allograft recipients during the first 8 weeks after transplantation. A total of 328 samples of resting (n = 251) and stimulated (n = 77, stimulated with phytohemagglutinin and lipopolysaccharide for 18 hours) peripheral blood mononuclear cells collected from 16 patients were measured. To measure intragraft cytokine levels, we analyzed 150 endomyocardial biopsy specimens from 19 patients. RESULTS: No elevation in expression was seen before injection, but, after the onset of rejection and concomitant with treatment, there was a decrease in detectable mRNA (p < 0.05) for the pro-inflammatory monokines interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha in resting peripheral blood mononuclear cells, and a decrease for the T-cell derived cytokines interleukin-4 and interleukin-10 in stimulated peripheral blood mononuclear cells. These changes in mRNA expression occurred coincidentally with decreases in the percentage of lymphocytes and monocytes in the peripheral blood after administration of rejection therapy. In endomyocardial biopsy specimens, there were no detectable changes in the quantities of cytokine mRNA specimens for the interferon-gamma, interleukin-6, interleukin-10, interleukin-1ra, and interleukin-1 beta genes before rejection. In general, the levels of these cytokines were near the lower limits of detection by our assay in endomyocardial biopsies, mRNA from the interleukin-2, interleukin-4, tumor necrosis factor-alpha, and interleukin-2R genes were undetectable. CONCLUSIONS: We conclude that changes in the expression of cytokine mRNA in both peripheral blood mononuclear cells and endomyocardial biopsy specimens as measured by the semiquantitative polymerase chain reaction method used in this study does not effectively predict rejection. The decline in peripheral blood mononuclear cell cytokine mRNA after rejection treatment is likely due to changes in the proportion of lymphocytes and monocytes in the peripheral blood in concert with a steroid-induced downregulation by cytokine gene transcription.

Donor specific bone marrow cells suppress lymphocyte reactivity to donor antigens and differentially modulate TH1 and TH2 cytokine gene expression in the responder cell population.

Previous studies have shown that post-transplantation infusion of donor specific bone marrow following a non-specific potent immunosuppressive agent such as antilymphocyte globulin (ALG) can significantly enhance graft survival compared to ALG alone. This enhancement remains variable and is thought to occur through the induction of specific partial tolerance to the renal allograft, but the underlying cellular mechanisms have not been clearly identified. In order to improve the efficacy of this specific immunosuppressive treatment and to study the events leading to enhanced allograft survival, we sought to establish a simple in vitro model based on a mixed lymphocyte reaction (MLR). We show that cellular proliferation seen in a normal MLR can be suppressed by addition of donor specific bone marrow cells (BMC). Significantly, this suppression is not observed with either third party BMC or donor specific peripheral blood mononuclear cells (PBMC). We have defined the optimum conditions of bone marrow infusion regarding number of BMC, their handling and culture, and simple enrichment procedures. Using a semiquantitative polymerase chain reaction assay, we have studied the cytokine gene expression in MLR modulated by donor specific BMC. In an unmodified allogeneic response, the responder cells show increased expression of interleukin-2 (IL-2) gamma-interferon IFN-gamma and receptor (IL-2R) mRNA, and no IL-10 mRNA. When responder cells are cultured with BMC of the stimulator, there is a 256-fold decrease in IL-2 mRNA, and a 64-fold decrease in IFN-gamma and IL-2R mRNA. There is also a 64-fold increase in IL-10 mRNA. This effect is even more marked when the BMC are depleted of CD3+ cells. The kinetics of addition of donor specific BMC to the normal allogeneic MLR culture and specificity of the action of BMC are also elucidated. Our data suggest that the enhancement of graft survival observed with donor BMC may operate through decreased proliferation of reactive T cell clones (due to decreased IL-2/IL-2R) and suppressed monocyte functions (due to decreased IFN-gamma and increased IL-10 gene expression).

Expression of E-selectin messenger RNA and protein in rheumatoid arthritis.

OBJECTIVE: To examine the de novo synthesis and cellular distribution of the E-selectin adhesion molecule in synovial tissues obtained from patients with rheumatoid arthritis (RA). METHODS: Immunohistochemistry techniques combined with in situ hybridization were used to examine RA synovium. RESULTS: There were numerous endothelial cells positive for E-selectin and E-selectin messenger RNA in the RA synovial membranes. Moreover, E-selectin expression appeared to correlate with inflammatory activity. CONCLUSION: The strong vascular expression of E-selectin indicates an activation of endothelial cells in the recruitment of cells associated with the chronic inflammation of RA.

Peripheral blood chimerism in renal allograft recipients transfused with donor bone marrow.

Experimental studies have shown that administration of antilymphocyte serum combined with donor bone marrow cells can induce tolerance to allograft tissue. We have initially reported application of these protocols in clinical studies of cadaveric renal allograft recipients who were treated with MALG and donor-specific bone marrow cells. To evaluate the effectiveness of the donor marrow cells in the production of chimerism, a detection method based on 32P-incorporated PCR was established. The 32P PCR was utilized with primers specific for the HLA class II, VNTR (D17S5 and D1S111), and/or Y-chromosome genes to detect the presence of allogeneic chimerism in the recipients. Immediately posttransplant, 26.4% of marrow recipients demonstrated the presence of allogeneic chimerism prior to the marrow transfusion as did 18% in the untransfused controls. In transfused patients, chimerism was detected most frequently during the 1-3-month interval after marrow transfusion (65%), and then diminished to 50-56% at 3-12 months posttransfusion. In the control group the frequency of allogeneic chimerism was gradually decreased and was undetectable in the majority of the patients beyond 3 months posttransplant while marrow-transfused recipients were more likely to have chimeric cells detected consistently beyond 3 months. Rejection episodes were significantly effected by the presence of chimerism in the recipients. Of the transfused patients, 91.3% who demonstrated allogeneic chimerism were rejection-free as compared with 8.7% who experienced at least one rejection episode (P = 0.01). While the presence of allogeneic chimerism in the control group was correlated with rejection-free graft survival, this difference did not reach statistical significance.

IL-2, IL-4, and IFN-gamma gene expression versus secretion in superantigen-activated T cells. Distinct requirement for costimulatory signals through adhesion molecules.

In the complete absence of APCs staphylococcal superantigens induced IL-2, IL-4, IL-5, IFN-gamma, and IL-2R gene transcripts in both highly purified human T cells and FACs sorted CD4+ memory (CD45RA-) T cells. Secretion of IL-2, IL-4, and IFN-gamma, as well as DNA synthesis, on the other hand, required the presence of monocytes. At cytokine gene transcript level, three patterns of expression were noted after superantigen activation of T cells in the presence vs the absence of APC. mRNA levels for IL-2 were markedly up-regulated in the presence of monocytes, IL-4 and IFN-gamma transcripts increased only modestly, and IL-5 and IL-2R mRNA levels were unaffected. Blocking mAbs against LFA-1 and LFA-3 added to staphylococcal enterotoxin B (SEB)-activated cultures of T cells and autologous monocytes, reproducibly decreased both T cell proliferation and genetic expression of IL-2, IL-4, IL-5, and IL-2R, although having little or no effect on IFN-gamma transcripts. Further, under those conditions of blocking, secretion of IL-2 and IL-4 was dramatically decreased, whereas IFN-gamma secretion remained essentially unchanged. In contrast, LFA-1 and LFA-3 mAbs completely abrogated IFN-gamma secretion from PHA-activated T cell-monocyte mixtures, although having no inhibitory effect on T cell proliferation. These results indicate a characteristic and differential involvement of adhesion molecule-mediated signals in superantigen-induced T cell proliferation, differential cytokine gene expression, and cytokine secretion.

A standardized approach to PCR-based semiquantitation of multiple cytokine gene transcripts from small cell samples.

A simple, rapid, reproducible, and nonradioisotopic method for semiquantitative analysis of cytokine mRNAs based on polymerase chain reaction (PCR) is described. RNA isolated from 2.5 million cells has proven sufficient to perform semiquantitative analysis of mRNA for 10 different cytokines. By this approach accurate assessment of mRNA levels for multiple cytokines can be made from as little as 2 ml of blood or about 3 mg of biopsy material. Total cellular RNA is quantitatively recovered by guanidinium isothiocyanate-acid-phenol extraction of a constant number of cells. Further quantitation of RNA is unnecessary. Highly reproducible PCR product formation occurs after specific amplification of aliquots of reverse transcribed test RNA. The photographic image of the ethidium bromide-stained gel accurately reflects the amount of PCR product loaded, both densitometrically and visually. PCR product generation is not affected by the presence of carrier RNA. Thus quantitative recovery of total RNA is possible even from a very small number of cells. Similarly, presence of a large excess of nonspecific RNA from nonexpressing cells does not affect amplification of the specific mRNA under study. A linear relationship between mRNA frequency and PCR product formation is observed over a 256- to 512-fold range. The actual mRNA concentration for each cytokine varies depending on the relative abundance of mRNA for that cytokine relative to total RNA. By performing two amplification cycles (28 and 35) on undiluted and 10-fold diluted RNA samples, the range of detection linearity is extended over a 5000-fold difference in input RNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor recognition and lytic competence of IL-2-activated lymphocytes: regulation of both antibody-independent and -dependent cellular cytotoxicity via P75 IL-2 receptor.

Fc receptor-positive lymphocytes (FcR+) contain lymphokine-activated killer cell (LAK) precursors that in response to IL-2 develop potent antitumor cytotoxicity. These FcR+ cells are also capable of antibody-dependent cytotoxicity (ADCC), which can be detected using fresh human peripheral blood lymphocytes (PBL) directed to murine targets, however, PBL-mediated ADCC to human tumors usually is very low, requiring a stimulation of the PBL, which also can be accomplished with IL-2. Using human melanoma tumor target cells, with and without the 14G2a monoclonal antibody, we examined in parallel the role of p75 IL-2 receptor for regulation of the induction of both LAK and ADCC forms of antitumor cytotoxicity. Enrichment of FcR+ cells from fresh peripheral blood by elutriation and flow cytometry, followed by varying periods of IL-2 culture, revealed a differential kinetics of activation. ADCC was detectable after PBL exposure to IL-2 for as short as the 4 h cytotoxicity assay, while LAK activation required more than 24 h of exposure. Elimination of the FcR+ cells by magnetic bead depletion from large granular lymphocyte populations (LGL) resulted in a loss of both LAK and ADCC. Addition of antibody known to block the binding of IL-2 to the p75 molecule of the IL-2 receptor complex (Mik-beta 1) to activation cultures at zero time resulted in abrogation of both cytotoxicities. These results suggest that differentiation and maturation of the ADCC effectors occurs in response to IL-2 via the p75 molecule, as also does LAK activation.(ABSTRACT TRUNCATED AT 250 WORDS)