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We have hypothesized that the chromosomal instability observed in scleroderma patients and their family members may result from the loss of long stretches of the telomeric repeat which is found at the ends of all linear chromosomes. We examined the telomere lengths in scleroderma (SSc) patients (n = 43), their family members (n = 182) and in age-matched controls (n = 96) using restriction fragment length polymorphism (RFLP) and chemiluminescent labelled probes. The average loss of telomeric DNA in SSc patients and family members was found to be 3 kb when compared to the controls. This loss was not related to age or the duration of the disease. These results may reflect a genetic predisposition for chromosomal instability in these families, or exposure to a common environmental agent. A wide variety of common environmental agents are known to produce chromosomal aberrations; these include fungicides, pesticides, air pollutants and drugs. Scleroderma-like syndromes may be induced by some of these agents. [ABSTRACT FROM PUBLISHER]

Families with more than one case of scleroderma are unusual. Four families each with two members (in one case monozygotic twins) with scleroderma (systemic sclerosis, SSc) were identified. Clinical, immunogenetic and autoantibody studies were carried out. Multicase SSc families cited in the literature were reviewed. Each family pair shared cutaneous subset of disease severity, and SSc-associated autoantibody. HLA typing showed two pairs shared an HLA-DR allele associated with scleroderma (DR3 or DR5), while one also had alleles reported in association with their SSc-specific autoantibody. Review of dates and ages of onset suggested that the timing of onset of scleroderma is more likely to have an environmental trigger than to be encoded genetically. [ABSTRACT FROM PUBLISHER]

We identified maternal microchimerism by fluorescence in-situ hybridisation in magnetically-separated CD4 or CD8 peripheral blood cells of eight of nine male patients with juvenile idiopathic inflammatory myopathy, compared with two of nine healthy male controls. We also found maternal microchimerism in inflammatory lesions (one skin sample and nine muscle biopsy samples) of all ten patients examined, compared with two of ten biopsy samples from patients with other muscle disorders. These results suggest that maternal cells may be involved in the pathogenesis of juvenile idiopathic inflammatory myopathy. [ABSTRACT FROM AUTHOR]

A comprehensive investigation into the impact of spectral baseline on temperature prediction in natural marine water samples by Raman spectroscopy is presented. The origin of baseline signals is investigated using principal component analysis and phytoplankton cultures in laboratory experiments. Results indicate that fluorescence from photosynthetic pigments and dissolved organic matter may overlap with the Raman peak for 532 nm excitation and compromise the accuracy of temperature predictions. Two methods of spectral baseline correction in natural waters are evaluated: a traditional tilted baseline correction and a new correction by temperature marker values, with accuracies as high as ± 0.2°C being achieved in both cases.

The design and operation of a custom-built LIDAR-compatible, four-channel Raman spectrometer integrated to a 532 nm pulsed laser is presented. The multichannel design allowed for simultaneous collection of Raman photons at two spectral regions identified as highly sensitive to changes in water temperature. For each of these spectral bands, the signals having polarization parallel to (∥) and perpendicular to (⟂), the excitation polarization were collected. Four independent temperature markers were calculated from the Raman signals: two-colour(∥), two-colour(⟂), depolarization(A) and depolarization(B). A total of sixteen datasets were analysed for one ultrapure (Milli-Q) and three samples of natural water. Temperature accuracies of ±0.4 °C-±0.8 °C were achieved using the two-colour(∥) marker. When multiple linear regression models were constructed (linear combination) utilizing all simultaneously acquired temperature markers, improved accuracies of ±0.3 °C-±0.7 °C were achieved.

A detailed investigation into the use of Raman spectroscopy for determining water temperature is presented. The temperature dependence of unpolarized Raman spectra is evaluated numerically, and methods based on linear regression are used to determine the accuracy with which temperature can be obtained from Raman spectra. These methods were also used to inform the design and predict the performance of a two-channel Raman spectrometer, which can predict the temperature of mains supply water to an accuracy of ± 0.5 °C.

Objective: To determine the prevalence of specific autoantibodies in a cohort of patients with systemic sclerosis (SSc) and a diagnosis of cancer, and to compare it to that in a group of patients with SSc who were never diagnosed with cancer., Methods: From 769 patients with SSc followed at our center over the past 15 years, 77 had a diagnosis of cancer. The results of autoantibody studies in this group were compared to those from 159 SSc patients without cancer randomly selected from the rest of the patient population using chi-square test for independence and the null hypothesis for 2 population proportions., Results: There was no statistically significant difference between the proportions of patients with positive autoantibodies between the 2 groups, except that a higher prevalence of autoantibodies with a nucleolar immunofluorescence pattern was observed in the group of patients who were never diagnosed with cancer (p < 0.01)., Conclusion: In contrast to previous studies, in our case-control study we were not able to detect a significant difference in autoantibody frequency or patterns among SSc patients with and without a diagnosis of cancer. These results refute the conclusion made previously that certain autoantibodies may represent risk factors for the development of cancer in patients with SSc.

Objective: To develop a murine model for use in examining the role of microchimeric cells and certain chemical exposures in the pathogenesis of systemic sclerosis (SSc)., Methods: Female BALB/cJ retired breeder mice were bled before and after vinyl chloride injection. The DNA from their white blood cells was obtained, and the number of microchimeric cell equivalents was determined by quantitative polymerase chain reaction using DNA primers specific for the H-2Kb gene, a sequence not found in BALB/cJ mice. Skin was obtained at autopsy, embedded in paraffin, sectioned, and stained with Masson's trichrome. Hydroxyproline analyses were performed on 4-mm skin biopsy samples., Results: Microchimeric cells were identified and quantitated before and after 20 daily intraperitoneal injections of vinyl chloride. The number of microchimeric cells in the peripheral blood increased an average of 48-fold after treatment with vinyl chloride. Histologic examination of the skin of these same mice (which had an increased number of microchimeric cells) showed inflammation, with abundant fibroblasts and a heavy mononuclear infiltration in the dermis. The collagen fibers appeared densely packed and disorganized. Histologic examination of the skin of untreated retired breeder mice and treated virgin mice appeared normal. Quantitative assays to determine the collagen content of skin biopsy samples obtained from treated microchimeric mice compared with nontreated microchimeric or with treated nonmicrochimeric mice showed a 2-3-fold increase in collagen content in the treated microchimeric mice. Extraordinary splenomegaly was present in the vinyl chloride-treated microchimeric mice, accompanied by cellular infiltration and fibrosis., Conclusion: The results suggest that vinyl chloride injections into BALB/cJ retired breeder mice lead to activation of microchimeric cells, which causes the cells to divide and multiply. The correlation between the 48-fold increase in microchimeric cells and the appearance of dermal inflammation and fibrosis similar to that of graft-versus-host disease suggests that activated microchimeric cells may be a necessary factor in the pathogenesis of autoimmune diseases such as SSc.

Objective: Systemic sclerosis (SSc; scleroderma), a disease of unknown origin, displays many similarities to chronic graft-versus-host disease. It occurs most frequently in women after the childbearing years. In recent studies, the presence of Y-chromosome DNA derived from male fetuses was detected, but DNA from female fetuses could not be investigated in those studies. The present study was undertaken to investigate cellular microchimerism of either male or female origin in DNA from patients with SSc., Methods: DNA from peripheral blood cells of 63 patients with SSc, 64 healthy individuals, and 24 non-SSc disease controls was examined by polymerase chain reaction analysis of HLA-Cw antigens., Results: Cellular microchimerism of either male or female origin was detected in 41 of 63 SSc patients (65%), in contrast to 18 of 64 normal controls (28%) (chi2 = 15.98, Pcorr = 0.00006) and 8 of 24 disease controls (33%)(chi2= 5.89, Pcorr, = 0.015)., Conclusion: These findings support the hypothesis that microchimeric cells persisting in the circulation or tissues of SSc patients could be involved in disease pathogenesis by initiating a graft-versus-host-like reaction.

Systemic sclerosis (SSc) is a disease of unknown origin. Although SSc is considered to be an autoimmune disease, recent studies have implicated cellular microchimerism in its pathogenesis. Microchimerism results from the persistence of fetal cells, from prior pregnancies, in the maternal circulation. The demonstration of the presence of fetal CD3+ T cells in the maternal circulation and of fetal cells in affected SSc tissues suggests that microchimerism might cause SSc in certain patients by initiating a graft-versus-host-like response.

Objective: To identify regulatory elements in the promoter region of the alpha1(I) procollagen gene (COL1A1) involved in the transcriptional activation of this gene in systemic sclerosis (SSc), and to identify the transcription factors interacting with these regulatory elements., Methods: Dermal fibroblasts from 6 patients with diffuse SSc of recent onset and from 6 healthy individuals were studied. The transcriptional regulation of COL1A1 was examined by transient transfections with deletion constructs containing portions of the COL1A1 promoter. The DNA binding activity of nuclear proteins recognizing the regulatory regions in the COL1A1 promoter was examined by gel mobility shift assays. A procedure was established to allow the quantitative determination of the amount of DNA binding proteins interacting with the COL1A1 promoter, employing DNA binding protein and DNA titration experiments analyzed by gel mobility shift assays., Results: Maximal chloramphenicol acetyltransferase activity was observed with a -174-bp to +42-bp COL1A1 promoter construct in both normal and SSc cells; however, the activity driven by this construct was 70-260% higher in SSc fibroblasts. Most of the transcriptional activity of the COL1A1 promoter was contained in a minimal promoter region encompassing -174 bp to -84 bp. Electrophoretic mobility shift assays performed with oligonucleotides corresponding to the regions spanning -129/-107 bp and -104/-78 bp of the COL1A1 promoter revealed marked increases in the intensities of DNA-protein complexes formed with both oligonucleotides in nuclear extracts prepared from each of the SSc cell lines in comparison with normal fibroblasts. Competition experiments showed that each of these regions contained elements recognized by Sp1 and nuclear factor 1 (NF-1) binding proteins. A quantitative determination of DNA binding activity recognizing the Sp1 binding element within the -129/-107-bp region showed that it was 23.6 nM in SSc fibroblasts compared with 6.9 nM in normal fibroblasts., Conclusion: The results demonstrate that a short region in the proximal promoter of COL1A1 containing 2 tandem NF-1/Sp1 elements displays up-regulated transcriptional activity in SSc fibroblasts, and that SSc fibroblasts contain 3.4-fold greater DNA binding activity recognizing these elements than normal cells.

We previously demonstrated that a segment of the human alpha1 type I procollagen gene (COL1A1) promoter encompassing nt -174 to -84 is responsible for the highest transcriptional activity in collagen producing cells in vitro. Here, we identified two almost identical tandem NF-1/Sp1 binding sites located between nt -129 to -107 (distal element) and nt -104 to -77 (proximal element) that are responsible for the basal regulation of COL1A1 transcription in normal human dermal fibroblasts. Transient transfection studies revealed that 85% of the basal COL1A1 promoter activity resides within the distal element; however, site-directed mutagenesis within the CCAAT motif in the proximal element resulted in a 98% decrease of the COL1A1 promoter activity. We conclude that each of the NF-1/Sp1 tandem binding sites has a different function. The distal element drives the transcriptional activity of the COL1A1 promoter but is not sufficient for its basal expression, whereas the NF-1 binding site in the proximal element is essential for in vitro COL1A1 gene transcription.

In previous work, we delineated a proximal region of the human alpha1(I) collagen gene (COL1A1) promoter necessary to direct its basal transcription in fibroblasts. This region has potential recognition sites for a variety of DNA-binding proteins. Here, we show that the -129/-107-bp sequence in this region of the promoter, which harbors an inverted CCAAT motif closely linked to a GC-rich direct repeat and is perfectly conserved between mouse and human, specifically bound the transcription factors Sp1, Sp3, and CTF/NF-1 in nuclear extracts from human skin and lung fibroblasts. Drosophila Schneider L2 cells lacking endogenous Sp activity were used to investigate the effect of expression of Sp or CTF/NF-1 transcription factors on COLlAl promoter activity. Expression of Sp1 caused potent trans-activation of a COL1A1 promoter (-174 to +42bp). In contrast, expression of Sp3, which binds to the same recognition sites as Sp1, and CTF/NF-1 stimulated COL1A1 promoter activity only at higher concentrations, and Sp2 did not transactivate. Expression of a 10-fold excess of Sp3, but not CTF/NF-1 or Sp2, abrogated the stimulation of COL1A1 promoter activity induced by Sp1. TGF-beta at concentrations previously shown to increase COL1A1 transcription caused a decrease in the relative amount of Sp3 in fibroblast nuclear extracts. These results suggest that both Sp1 and Sp3 bind to the proximal COL1A1 promoter and stimulate its activity; however, their interaction with each other may result in repression of Sp1-induced COL1A1 transcription. Alterations in the relative amounts or DNA-binding activities of these transcription factors in a cell- or signal-specific manner may contribute to the control of transcription from the COL1A1 promoter. The present findings, and recent observations implicating Sp1 and its homologs in regulating the expression of several collagen genes, suggest that the family of Sp1 transcription factors play a role in physiological and pathological modulation of connective tissue accumulation.

Background: Systemic sclerosis is a disease of unknown origin which often occurs in women after their childbearing years. It has many clinical and histopathological similarities to chronic graft-versus-host disease. Recent studies indicate that fetal stem cells can survive in the maternal circulation for many years post partum. This finding suggests that fetal cells persisting in the maternal circulation or tissues could be involved in the pathogenesis of systemic sclerosis by initiating a graft-versus-host reaction., Methods: We used the polymerase chain reaction (PCR) to identify Y-chromosome sequences in DNA extracted from peripheral-blood cells and skin lesions from women with systemic sclerosis of recent onset. To confirm the PCR findings, we used fluorescence in situ hybridization of peripheral-blood cells and cells within chronic inflammatory-cell infiltrates in biopsy specimens of affected skin., Results: Y-chromosome sequences were found in DNA from peripheral-blood cells in 32 of 69 women with systemic sclerosis (46 percent), as compared with 1 of 25 normal women (4 percent, P<0.001), and in T lymphocytes from 3 women with systemic sclerosis who had male offspring. Furthermore, Y-chromosome sequences were identified in skin-biopsy specimens from 11 of 19 women with systemic sclerosis (58 percent); 9 of the 11 were known to have carried male fetuses. Nucleated cells containing Y chromosomes were detected by fluorescence in situ hybridization in paraffin-embedded sections of skin lesions from all seven women we tested whose skin-biopsy specimens contained Y-chromosome sequences., Conclusions: Fetal antimaternal graft-versus-host reactions may be involved in the pathogenesis of systemic sclerosis in some women.

Systemic sclerosis (SSc) is a disease of unknown origin, which occurs predominantly in women after childbearing years. There are prominent clinical and histopathologic similarities between SSc and chronic graft-versus-host disease (GVHD). GVHD can occur after blood transfusions or after transplantation with HLA-compatible bone marrow. Here we examined the hypothesis that SSc may be caused by fetal cells crossing the placenta into the maternal circulation and providing donor lymphocytes which recognize disparate HLA antigens, resulting in a reaction similar to chronic GVHD. To test the hypothesis we analyzed the inheritance of HLA class I and class II haplotypes in the families of 37 SSc patients and 42 control individuals. Twenty-six (70.2%) SSc patients had HLA class II alleles compatible either for their offspring or mother, compared with only nine (21%) control individuals. The four patients with juvenile onset SSc we analyzed had alleles compatible with their mothers. These results suggest that in some patients, SSc may, indeed, be a form of chronic GVHD caused by fetal or maternal cells which have crossed the placenta during pregnancy and have remained unrecognized by the host due to class II HLA compatibility, and that subsequent activation of these cells by as yet unknown stimuli result in the development of the disease.

We have characterized genetic alterations at the molecular level in 49 scleroderma and 45 control families using variable number tandem repeats (VNTRs). Additionally, paired fibroblast cell lines from the 'affected' and 'unaffected' skin and peripheral blood leucocytes of 30 patients were also examined. All families in this study were typed for Class I Cw alleles and Class II-DRB, -DQA and -DQB to confirm family membership. There were significant rises in the level of VNTR mutations in scleroderma patients (36.7%, n = 18), their siblings (16.3%, n = 13) and offspring (21.7%, n = 15). The level of VNTR mutations in the control group was 0.6% (n = 5). These mutations did not correlate with the presence of autoantibodies and no patient was taking a known clastogenic drug. The most common VNTR site for mutation was pYNZ22 (17p13.4). Differences were also seen in the VNTR alleles between fibroblast and lymphocyte DNA from the same patient, as measured by size alteration of one of the alleles. We have found that VNTRs are unstable in scleroderma patients, relatives and offspring. The reason for the genomic changes remains unknown, but previous studies have implicated the presence of a clastogen.

Analysis of the regulatory promoter region of the human alpha 1 (I) collagen-encoding gene (COL1A1) gene indicated the presence of G+C-rich sequence elements that are potential binding sites for the transcription factor Sp1. As a step toward understanding transcriptional regulation of the human COL1A1, we examined Sp1 binding in the promoter region using DNase I footprinting, and analyzed the effect of Sp1 expression on COL1A1 promoter activity in transiently transfected Drosophila melanogaster cells in vivo. The results indicated that recombinant human Sp1 interacted specifically with two G+C-rich sequences within the COL1A1 promoter. Binding of factors in nuclear extracts prepared from human dermal fibroblasts to a 22-nucleotide deoxyribonucleotide (oligo) spanning the 5' G+C-rich sequence required Zn2+, and was abolished by excess Sp1 consensus binding site oligos, or by anti-Sp1 antibodies. Studies in which a series of progressively 5'-deleted COL1A1 promoter::cat constructs were co-expressed with an Sp1 expression plasmid in a cellular background devoid of Sp1 homology demonstrated that Sp1 markedly enhanced the COL1A1 promoter activity. These results suggest that the transcriptional activity of the human COL1A1 can be positively regulated by Sp1.

The frequency and functional properties of anti-topoisomerase-1 antibodies (ATA) have been studied in 58 systemic sclerosis (SSc) probands, 218 first degree relatives and 22 spouses. The dependence of ATA on the presence of certain HLA-DRB1 and HLA-DQB1 alleles was examined. ATA were detected by immunodiffusion, by absorption or inhibition of topoisomerase-1 enzymic activity, by immunoblotting of a K562 cell extract and by immunoprecipitation of 35S radiolabelled cell lines. HLA class II typing for HLA-DRB1 and HLA-DQB1 was performed by oligonucleotide typing in 49 families. Six probands and two relatives had ATA. The relatives with ATA had SSc. All eight individuals with ATA directly inhibited topoisomerase-1 function. Four of the eight had limited skin disease and four had diffuse skin involvement. The seven who were genotyped had at least one HLA-DQB1 allele encoding for tyrosine at position 30 of the first domain. Therefore, ATA are not widely dispersed within families, but rather are only present in those with SSc, and certain genetic requirements appear necessary for their generation.

Background: Herpes gestationis (HG) is a rare, pregnancy-related skin disease characterized by the production of an autoantibody to a component of the hemidesmosome. It is associated with the class II antigens HLA-DR3 and HLA-DR4, but its potential association with the "class III antigens" C2, C4, and factor B has not previously been studied., Objective: Our purpose was to study complement polymorphism in HG., Methods: Using electrophoresis and immunofixation techniques, we determined the allele frequencies of C4A, C4B, C3, and factor B in 42 patients with a history of HG., Results: Ninety percent of patients carried a C4 null allele (C4*QO). No statistically significant association with C3 or factor B alleles was seen., Conclusion: HG is associated with the presence of a C4*QO. Whether the C4*QO is the primary genetic association, or whether the C4*QO is related to its linkage disequilibrium with DR3 and DR4 has yet to be determined.