Excess BMP Signaling in Heterotopic Cartilage Forming in Prg4-null TMJ Discs.

Heterotopic cartilage develops in certain pathologic conditions, including those affecting the human temporomandibular joint (TMJ), but the underlying molecular mechanisms remain obscure. This is in part due to the fact that a reliable animal model of such TMJ diseases is not available. Here, we show that aberrant chondrocyte differentiation and ectopic cartilage formation occur spontaneously in proteoglycan 4 (Prg4) mutant TMJ discs without further invasive procedure. By 2 mo of age, mutant disc cells displayed chondrocyte transdifferentiation, accompanied by strong expression of cartilage master gene Sox9 and matrix genes aggrecan and type II collagen. By 6 mo, heterotopic cartilage had formed in the discs and expressed cartilage hypertrophic markers Runx2 and ColX. The ectopic tissue grew in size over time and exhibited regional mineralization by 12 mo. Bone morphogenetic protein (BMP) signaling was activated with the ectopic chondrogenic cells and chondrocytes, as indicated by phosphorylated Smad 1/5/8 nuclear staining and by elevated expression of Bmp2, Bmpr1b, Bmpr2, and BMP signaling target genes. Likewise, we found that upon treatment with recombinant human BMP 2 in high-density micromass culture, mutant disc cells differentiated into chondrocytes and synthesized cartilage matrix more robustly than control cells. Importantly, a specific kinase inhibitor of BMP receptors drastically attenuated chondrogenesis in recombinant human BMP 2-treated mutant disc cultures. Unexpectedly, we found that Prg4 was expressed at joint-associated sites, including disc/muscle insertion and muscle/bone interface, and all these structures were abnormal in Prg4 mutants. Our data indicate that Prg4 is needed for TMJ disc integrity and function and that its absence leads to ectopic chondrogenesis and cartilage formation in conjunction with abnormal BMP signaling. Our findings imply that the BMP signaling pathway could be a potential therapeutic target for prevention or inhibition of ectopic cartilage formation in TMJ disease.

Copper(i)oxide microparticles - synthesis and antimicrobial finishing of textiles.

Copper containing particles are of high interest to provide antibacterial activity to textiles for medical products, hygiene application or where odor formation as result of bacterial activity has to be controlled. Cu(i)oxide microparticles with a rather uniform diameter between 1.5 and 2 µm can be prepared by controlled reduction of alkaline Cu(ii)-tartaric acid complexes. Such particles can be bound to textile surfaces by means of a pigment binder system used in pigment dyeing. By a simple pad-dry process textile fabrics with a Cu-content of 250-270 mg Cu per kg fabric could be prepared. The samples (fabrics) exhibited a reduction in viability of 100% for Staphylococcus aureus and 84% for Klebsiella pneumonia as estimated by the ASTM E2149 antimicrobial test. Simulated wash procedures led to a reduction in Cu-content to 60-50% of the initial value. Reduction in viability remained at 99% for Staphylococcus aureus and 78% for Klebsiella pneumoniae. The new process is of high value to impart antimicrobial properties to textile products because an antimicrobial product with good wash permanence can be delivered using rather simple processing and ordinary chemicals.

In vivo effectiveness of enamel sealants around orthodontic brackets.

OBJECTIVE: In this randomized study, the caries-protective effect on vestibular enamel of two fluoride-containing sealants (Protecto® and Light Bond®) during multibracket treatment was investigated. MATERIALS AND METHODS: In all, 40 orthodontic patients about to receive a multibracket appliance with the brackets bonded to the vestibular tooth surfaces were randomly included in this study. Each one was randomly assigned to one of four groups. A crossover design was selected in which a sealed quadrant was contralateral to an unsealed quadrant, then choosing the reverse configuration in the opposite jaw. Two sealants were, thus, tested on vestibular enamel on left and right anterior teeth and premolars in both jaws of each patient over 6 months of multibracket treatment. A DIAGNOdent® pen measuring laser fluorescence was used to analyze the relevant enamel surfaces both at baseline and after 6 months. RESULTS: Neither the incidence nor the characteristics of the demineralization we observed during the study differed between the 4 groups. CONCLUSION: Single application of smooth-surface sealants did not protect enamel around brackets from incipient carious lesions during the first 6 months of multibracket treatment.

Integration of a maxillary model into facial surface stereophotogrammetry.

AIMS: Three-dimensional (3D) integration of a maxillary model into a facial model has only been possible by a complex procedure using face bow transfer after taking impressions of certain maxillary and facial parts. In this study, we aimed to develop a method for integrating a scanned maxillary model into a scan-realized facial model. MATERIAL AND METHODS: A total of 19 patients with the medical indication for cone-beam computed tomography (CBCT) and orthodontic treatment were included in this study. Facial and maxillary scans were also taken. The construction of the integrated surface model required 10 steps. This integration procedure was evaluated by taking ten 3D dentofacial linear segment measurements in the integrated scan and the CBCT. These results were analyzed using descriptive statistics. RESULTS: All measurements demonstrated good intra-individual reliability. We observed almost perfect congruence between integrated scan and CBCT in vertical distances, while the sagittal measurements revealed more, yet clinically acceptable, deviations possibly caused by different error sources in either of the two methods. CONCLUSION: This new method is suitable for generating 3D integrated surface-scan models which can be used for growth and therapy control studies in orthodontics and other disciplines in the dentofacial fields. Since this method does not require ionizing radiation, it is highly recommendable as an application for children and adolescent patients.

Process balance and product quality in the production of natural indigo from Polygonum tinctorium Ait. applying low-technology methods.

Indigo is the most important blue component in the class of natural dyes for cellulose and protein fibres. In the moderate European climate Polygonum tinctorium Ait. could be an interesting source for natural indigo (Vat blue 1). Following a cultivation of the plant material a simple procedure for the extraction of the indigo precursor indican was investigated with regard to crop and quality of dye obtained. The dependence of the crop on the storage conditions of the harvested plant material was investigated. The results quantify the distinct sensitivity of the fresh material to the time of storage before extraction with regard to the amount of natural indigo obtained, the photometrically determined indigo content in the product and the shade and colour depth observed in standardised dyeing experiments. A basic set of data is presented, which describes the process in terms of consumption of energy, water and chemicals and organic waste released from the extraction step.

Differential stem cell contributions to thymocyte succession during development of Xenopus laevis.

The contribution of two embryonic stem cell compartments to the developing thymus in the amphibian Xenopus was examined throughout the larval, postmetamorphic, and adult periods. Hematopoietic chimeras were produced by transplanting either the ventral blood islands (VBI) or the dorsal stem cell compartment (DSC) from diploid donors onto triploid hosts. The DNA content of isolated nuclei harvested from the thymus and circulating E populations was analyzed using propidium iodide staining and flow cytometry. The DNA content of mitotic figures derived from PHA reactive splenocytes was analyzed using the Feulgen reaction and microdensitometry. These data suggested that both the VBI and DSC contribute to the thymocyte populations from the earliest developmental stages examined. Moreover, the contribution of both stem cell compartments was cyclic. However, the periods of these cycles were different. Both VBI- and DSC-derived cells entered the thymus 4 days postfertilization. VBI-derived thymocytes were at a minimum at 28 days postfertilization, reached a maximum at 35 days postfertilization and a second minimum at 42 days postfertilization. However, DSC-derived cells reached a maximum at 28 days, a minimum at 35 days, and a second maximum at 42 days. The PHA-reactive splenocyte population followed a similar temporal pattern. In contrast, the VBI-derived E population was at a maximum during early development and steadily declined throughout the larval period. DSC-derived E were undetectable during early development but steadily increased throughout the larval period. Both VBI- and DSC-derived hematopoietic cells persisted after metamorphosis and contributed to all populations examined in adult frogs. Because of temporal differences in the VBI and DSC contributions to the developing thymus, these data suggest heterogeneity within the thymocyte population associated with the embryonic origin of the colonizing stem cells.

In-vitro infection of chronic lymphocytic leukemia cells by Epstein-Barr virus (EBV).

We sought to determine the potential of infecting lymphoid cells from patients with chronic leukemia (CLL) with Epstein-Barr virus (EBV) by testing for EBV receptors (EBVR) by flow cytometry, assessing for infectability of these cells by culturing with B95-8-derived virus, and staining for EB nuclear-associated antigens (EBNA) at various times post-infection. EBVR were present on 54-91% of lymphoid cells in seven cases of CLL and on 46% of prolymphocytic leukemia cells. Dynamic changes regarding EBNA positivity, morphology, and viability occurred post-infection with the virus. On day 2 only a few EBNA-positive lymphoblasts were observed. On days 11-21 positivity increased from 2 to 34% of cells. Simultaneously, the viable cell number declined to approximately 1/10th of original number. A significant proportion of the EBNA-positive cells corresponded to the original CLL cells. In 3 of 7 cases of CLL a Pan T-cell phenotype was demonstrated by Leu-1 monoclonal antibody testing. The infected cells did not react with two monoclonal antibodies, EBV-CS 1 and 4, which react with B-cell lymphoblastoid cell lines (B-LCL). Moreover, the B-LCL derived at 1-2 months post-infection of CLL cells did not express the Leu-1 antigen, but expressed EBV-CS 1 or 4 defined antigens. In the prolymphocytic leukemia, 64% of the cells showed EBNA positivity on day 7 and giant cells with huge round or multiple nuclei appeared which were EBNA-positive. CLL and prolymphocytic leukemia cells can be infected as demonstrated by EBNA-positivity. This infection does not lead to immediate transformation, but evokes lymphoblast and multinucleated giant cell production prior to the death of cells.

A method for determination of antibody-dependent cellular cytotoxicity (ADCC) of human peripheral mononuclear cells.

A method is described for measuring antibody-dependent cellular cytotoxicity (ADCC) of mononuclear cells from human peripheral blood using an established murine cell line and commercially prepared antisera. The test utilizes a standard 51Cr release technique. The ADCC activity of mononuclear cells obtained from 10 healthy human volunteers was measured at 4 different effector: target cell ratios. A linear relationship between %51Cr release (ADCC) and the number of effector cells was observed.

Chronic mononucleosis syndrome.

We present data on 14 patients with chronic symptoms of disabling fatigue in association with serologic evidence of active Epstein-Barr virus (EBV) infection. Two thirds were women, and the average age at onset was 29.6 years. Forty-three percent were known to have had previous infectious mononucleosis, but the usual criteria for that diagnosis were not helpful with the present syndrome. Eighty-six percent had serologic evidence of cytomegalovirus (CMV) infection. Profound immunodeficiency was not present, but 71% had partial hypogammaglobulinemia, and minor abnormalities of T cell subsets were noted in six of seven patients studied. Fifty-seven percent achieved temporary serologic and symptomatic remission after an average duration of 33 months. Only one patient has a sustained remission. Comparison is made with other reported chronic, recurrent, and persistent EBV syndromes, and tentative diagnostic criteria for chronic mononucleosis syndrome are presented. Recently available EBV serologic techniques allow for identification of patients who have reactivated EBV infection, and this reactivation may be related to symptoms.

Delayed onset of infectious mononucleosis associated with acquired agammaglobulinemia and red cell aplasia.

In 1974, an 11-year-old white boy with the X-linked lymphoproliferative syndrome developed hyper-IgM after becoming infected with Epstein-Barr virus. However, he failed to develop normal immune responses against the virus. In December 1981, when red cell aplasia occurred, he was given packed erythrocytes and gammaglobulin. Nine weeks later, acute infectious mononucleosis developed. Concurrently, his T4/T8 helper/suppressor ratio decreased from 2.7 to 0.2, and IgM antibodies to Epstein-Barr virus appeared. Subsequently, circulating B cells became undetectable in his blood, and agammaglobulinemia appeared. Red cell aplasia abated transiently. This patient's course was complicated by Haemophilus influenzae and Mycobacterium tuberculosis pneumonias, and red cell aplasia and agammaglobulinemia have persisted. Epstein-Barr virus acting as a slow virus probably induced the red cell aplasia and agammaglobulinemia because of the aberrant immune responses to Epstein-Barr virus. Immunodeficient responses to Epstein-Barr virus should be sought in other patients with the diseases documented in our patient.

Epstein-Barr virus and chronic lymphadenomegaly in male homosexuals with acquired immunodeficiency syndrome (AIDS).

Male homosexuals at risk for developing AIDS frequently exhibit chronic lymphadenomegaly (LAD). They are at high risk for developing malignant B cell lymphomas. A study of Epstein-Barr virus (EBV) revealed marked abnormalities in these patients. One hundred percent of the patients were seropositive. The patients with most severe acquired immune deficiency disorders manifested a decreased number of circulating B cells with EBV receptors and decreased lymphocyte transformation. Patients often showed defective memory T cell cytotoxic responses to autologous EBV infection in vitro. Three of five lymph node specimens contain significant EBV genome copies to suggest a significant etiologic role. In addition, a Burkitt-like lymphoma carried EBV genome. Although all of the men were seropositive for EBV, reactivation patterns were not as common as anticipated. Given the presence of EBV genome in the lymph nodes of the patients who lack anti-early antigen (EA) antibodies indicative of reactivation, we suggest that reliance on serology to indicate EBV involvement is insufficient for assessing the patient. The detection of a t(8;14) transposition in the monoclonal mu kappa Burkitt-like lymphoma containing EBV genome supports the view that cytogenetic transposition is a mechanism in lymphomagenesis.

Lymphocyte abnormalities in preleukemia--I. Decreased NK activity, anomalous immunoregulatory cell subsets and deficient EBV receptors.

The development of acute non-lymphocytic leukemia is preceded by a set of symptoms described as the preleukemia syndrome. Six patients who fulfilled the criteria for this preleukemia syndrome have been evaluated for abnormalities in the lymphocyte population. The NK cell activity was reduced, the immunoregulatory cell populations were numerically abnormal, and the B cell subpopulation was deficient in EBV receptors. Thus, in addition to the abnormalities in the myeloid populations, there are serious defects in the lymphoid systems of preleukemic patients.

Comparison of ADCC and NK activities of peripheral blood mononuclear cells from patients at risk for acquired immune deficiency syndrome (AIDS).

Numerous cellular immune defects have been described in patients with acquired immune deficiency syndrome (AIDS). These include anergy, reduced numbers of helper T cells, and decreased effectiveness of natural killer (NK) cells. In this study, we have measured the lytic activity of antibody-dependent cellular cytotoxic (K) cells using a recently described 51Cr release assay in patients clinically displaying lymphadenopathy syndrome (LAD). We then compared ADCC activity with NK activity, T helper/T suppressor ratios and NK cell number in these same patients. Our results indicate that a reduction of ADCC activity can occur in patients with LAD. In our study, patients that showed reduced ADCC activity also showed reduced NK cell function. Other individuals in this study had reduced NK function while ADCC values remained within the normal range. The degree of reversal of the T helper/T suppressor cell ratio was not a useful indicator of cellular immune function as measured by these assays in that individuals with very low ratios often had normal cytotoxic cell function while some patients with ratios in the normal range had decreased cytotoxicity. The percentage of NK cells as determined by HNK-1 monoclonal antibody also did not prove useful in defining potential cytotoxic activities.

Immune deficiency in the X-linked lymphoproliferative syndrome. I. Epstein-Barr virus-specific defects.

Eleven males with XLP were evaluated for EBV-specific antibodies during periods of 2 to 7 yr. Variable responses to EBV-specific antigens were found. All 11 patients had subnormal anti-EBNA titers, which probably reflected a T cell deficiency. The patients showed four different patterns in their anti-VCA response: 1) two boys who had experienced malignant lymphoma mounted no antibodies at all; 2) two patients showed intermittent anti-VCA titers; 3) four males had persistently elevated anti-VCA titers; and 4) three patients showed normal anti-VCA titers. ADCC against EBV-infected cells was abnormally low in six patients and was elevated in two patients given gamma-globulin. ADCC titers did not correlate with anti-VCA titers. However, most patients with XLP failed to effect regression of autologous EBV-infected lymphoblastoid cell lines, indicating a deficiency in long-lived T cell-mediated immunity to EBV.

Cell-mediated immunity to Epstein-Barr virus (EBV) and natural killer (NK)-cell activity in the X-linked lymphoproliferative syndrome.

The activity of T-cell-mediated immunity to Epstein-Barr virus (EBV) was assessed by an assay of regression of the outgrowth of EBV-infected autologous B cells. Regression and natural killer (NK)-cell activities were compared for patients and their mothers from five families with X-linked lymphoproliferative syndrome (XLP) and three control groups. Seven of the 10 patients with XLP exhibited weak T-cell activity against autologous EBV-infected lymphoblastoid cell lines (LCL) comparable to EBV-seronegative controls. In contrast, 8 of 10 obligate carrier females of XLP had unusually strong activity, which was comparable to anti-early-antigen (EA) positive controls. The results of the regression assays correlated with their EBV serology: mothers showed high titers, and their affected sons showed low-titer EBV-specific antibody responses. Defective NK-cell activity was found only in the patients with XLP. NK and regression activities did not correlate. Our findings explain, in part, the vulnerability to EBV of males with XLP and why their mothers are protected from life-threatening phenotypes of XLP.

Epstein-Barr virus in normal pregnant women.

Acquired immune suppression accompanying normal pregnancy may be associated with reactivation of Epstein-Barr virus (EBV). Pregnant women with reactivated EBV having anti-EA antibodies show high titers of antiviral capsid antigen (VCA) geometric mean titers (GMT) of 522 versus 170 in those lacking anti-early antigen (EA). Among twenty-seven seropositive women at parturition, 17 (63%) had generated antibody to EA, and all 27 (100%) demonstrated significant increases in antibody to VCA (p less than 0.01). In contrast, antibody titers to cytomegalovirus, herpes hominis, varicella-zoster, and rubella viruses in the pregnant women were comparable to those found in nonpregnant controls. (Am J Reprod Immunol. 1982; 2:217-221.)

Epstein-Barr virus-induced diseases in boys with the X-linked lymphoproliferative syndrome (XLP): update on studies of the registry.

Analyses of 100 subjects with the X-linked lymphoproliferative syndrome (XLP) in 25 kindreds revealed four major interrelated phenotypes: infectious mononucleosis, malignant B-cell lymphoma, aplastic anemia, and hypogammaglobulinemia. Eighty-one of the patients died. Two male subjects were asymptomatic but showed immunodeficiency to Epstein-Barr virus (EBV). Seventy-five subjects had the infectious mononucleosis phenotype and concurrently, 17 subjects of this group had aplastic anemia. All subjects with aplastic anemia died within a week. Aplastic anemia did not accompany hypogammaglobulinemia or malignant lymphoma phenotypes. Hypogammaglobulinemia had been detected before infectious mononucleosis in three subjects, after infectious mononucleosis in five subjects, and was not associated with infectious mononucleosis in 11 boys with hypogammaglobulinemia. In nine subjects infectious mononucleosis appeared to have evolved into malignant lymphoma; however, the majority of patients with malignant lymphoma showed no obvious antecedent infectious mononucleosis. One subject had infectious mononucleosis following recurrent malignant lymphoma. Twenty-six of 35 lymphomas were in the terminal ileum. Results of immunologic and virologic studies of 15 survivors revealed combined variable immunodeficiency and deficient antibody responses to EBV-specific antigens. Mothers of boys with XLP exhibited abnormally elevated titers of antibodies of EBV. Subjects of both sexes with phenotypes of XLP should be investigated for immunodeficiency to EBV. Persons with inherited or acquired immunodeficiency may be vulnerable to life-threatening EBV-induced diseases.

Construction of T cell hybridomas secreting allogeneic effect factor.

T cell hybridoma lines were constructed by fusion of DBA/2 alloantigen-activated T cell blasts with the AKR thymoma line BW5147. Certain of the hybridomas prepared in this manner secreted spontaneously into their culture supernates biologically active molecules that displayed B cell- and T cell-activating properties characteristic of allogeneic effect factor (AEF). Cell surface phenotype analysis documented that the hybridomas were, indeed, somatic cell hybrids between the two respective partner cells used for fusion. The B cell-activating properties of these hybridoma supernates was demonstrated by their capacity to stimulate T cell-depleted spleen cells to respond in vitro to T-dependent antigens. The T cell-activating properties of these hybridoma supernates was verified by their capacity to stimulate autonomous development of self-specific cytotoxic T lymphocytes and by their capacity to exert mitogenic effects on unprimed T cells. The biologically active molecules secreted by these hybridomas were, like conventional AEF, inhibitable by specific anti-Ia antibodies thus indicating the presence of Ia determinants on the relevant hybridoma products. Finally, these AEF-secreting hybridomas could be stimulated to proliferate and to secrete increased quantities of AEF when exposed to the specific alloantigen-bearing target cells to which the T cell blasts had been originally sensitized.

The biologic effects of allogeneic effect factor on T lymphocytes. I. The mitogenic activity and the autonomous induction of cytotoxic T lymphocytes by AEF.

Studies presented herein illustrate the capacity of the soluble mediator, allogeneic effect factor (AEF), which is derived from histoincompatible cell interactions, to induce the in vitro differentiation of normal murine splenic lymphocytes into mature cytotoxic cells capable of exerting activity on H-2-identical target cells. This process requires the presence of T lymphocytes during the sensitization phase, and the lytic activity on tumor cells is mediated by cytotoxic T lymphocytes (CTL). The capacity of AEF to induce differentiation of such CTL does not require the presence of stimulating target cells in the sensitization phase. The induction of CTL requires the presence of AEF at the initiation of culture, although exposure to AEF as brief as 1 hr is sufficient to induce fresh spleen cells to differentiate into CTL during the subsequent 5 days in culture. In addition to its ability to induce CTL, AEF is highly mitogenic for T lymphocytes. However, the mitogenic and the CTL-inducing activities of AEF can be experimentally dissociated, indicating that different subpopulations of T lymphocytes may be involved in the response to AEF. In contrast to similar soluble helper factors derived from allogeneic cell interactions, AEF appears to be unique in its ability to autonomously induce a primary CTL response in vitro.