1 alpha(OH)D3 (ETALPHA) treatment and receptor studies in 16 patients with chronic and myeloproliferative disorders

10 patients with CLL and 2 with CML were treated with gradually increasing doses of 1 alpha(OH)D3, up to 4 micrograms daily during 6 wk. 3 patients with preleukemia and 1 with myelofibrosis were treated with 2 micrograms daily of 1 alpha(OH)D3 for a prolonged period up to 17 wk. The treatment with 1 alpha (OH)D3 did not result in changes of disease parameters in any of the patients under study. Receptor studies for 1,25(OH)2D3 were performed in 8 CLL patients and revealed only 1 patient with increased specific receptor binding capacity. The maximum tolerable dose of 1 alpha(OH)D3 varied individually, but was in the range of 2-4 micrograms daily.     

Entactin: a possible auto-antigen in the pathogenesis of non-Goodpasture anti-GBM nephritis

It has recently been demonstrated that many patients with various types of glomerulonephritis have antibodies to the 6M guanidine-HCl extract of glomerular basement membrane (Bygren et al, Nephrol Dial Transplant 4:254-261, 1989). In the present study a 150 K protein was isolated from the guanidine extract of bovine glomerular basement membrane utilizing ion exchange and gel filtration chromatographic procedures. Amino acid analysis and size of the isolated protein revealed similarity to that of entactin/nidogen. The identity of this protein as entactin/nidogen was further suggested by its precipitation with two different antibodies in a radioimmunoassay and by its reaction with four different antibodies in a sandwich ELISA. Inhibition of the antibodies to 150 K by bovine entactin, which was isolated separately and sequenced for amino acids, confirmed the identity of the 150 K protein as entactin/nidogen. Furthermore, it was shown that about one third of those patients who show antibodies to the crude guanidine extract have circulating antibodies directed against entactin. This was further confirmed by the competitive inhibition of antibodies to the crude guanidine extract in one of the positive serum by entactin in an ELISA inhibition and by immunoblotting experiments. These observations propose entactin as a possible non-Goodpasture glomerular basement membrane antigen that could be involved in the pathogenesis of certain forms of autoimmune glomerulonephritis (non-Goodpasture anti-GBM glomerulonephritis) in man. Most of these patients have a granular pattern of the immunoglobulin deposition along the glomerular basement membrane. This suggests the possibility that anti-GBM glomerulonephritis in human beings can have non-linear immunoglobulin deposits along the GBM.     

An occipito-temporal syndrome in adolescents with optimally controlled hyperphenylalaninaemia

Summary The study included 16 adolescents with optimally controlled hyperphenylalaninaemia (McKusick 26160), of whom six did not require treatment according to conventional criteria. All except the two patients with lowest median serum phenylalanine level throughout childhood (most values at 200–300 µmol/L) had white matter abnormalities detectable with magnetic resonance imaging. The lesions were particularly prominent in the watershed regions between the posterior and middle cerebral arteries. In most patients with moderate or severe hyperphenylalaninaemia frontal white matter lesions were present as well. Normal proton magnetic resonance spectra indicated that the lesions were stable. Occipital EEG abnormalities were frequent, and deficient performance on a pattern-recognition test was a characteristic neuropsychological finding. Serum phenylalanine levels at about 300 µmol/L or below throughout childhood and early adolescence may be required to avoid lesions. The present study demonstrates the limitations of even an optimally controlled dietary regimen in hyperphenylalaninaemia.     

Glomerulonephritis induced in sheep by immunization with human glomerular basement membrane

The specificity of the anti-glomerular basement membrane (GBM) antibodies in experimental nephritis in sheep (Steblay's nephritis) was studied and compared with the specificity of antibodies in human anti-GBM nephritis (Goodpasture's syndrome). Sheep were injected monthly with isolated human GBM and antibody reactivities with isolated human and sheep GBM proteins were quantified with ELISA. Expectedly, the sheep had high titers of antibodies against several human GBM antigens. These antibodies remained for the most part in the circulation. In contrast, circulating antibody levels against sheep GBM antigens remained low for a long period of time, but a significant and progressive increase coincided with the development of acute nephritis. These antibodies accumulated in the kidneys of the nephritic sheep and could be eluted from diseased kidneys. They represent auto-antibodies immunologically cross-reacting with antigens of both sheep and human GBM. The specificity of auto-antibodies eluted from the kidneys was analyzed by immunoblotting and ELISA. The major populations reacted with one subunit, termed M2, of the globular domain of collagen IV. The same subunit contains the major antigen in Goodpasture's syndrome. It is concluded that the M2 subunit of the globular domain of collagen IV is recognized by IgG antibodies that primarily bind to the glomerular basement membrane in both Steblay's nephritis and Goodpasture's syndrome, indicating that it is a main nephritogen in both diseases.     

An ELISA for the detection of anti-neutrophil cytoplasm antibodies (ANCA)

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of circulating anti-neutrophil cytoplasm antibodies (ANCA), which are defined by a diffuse, granular staining of the cytoplasm of alcohol-fixed human neutrophils by indirect immunofluorescence (IIF). Detection of antineutrophil cytoplasm antibodies has a high sensitivity and specificity for active Wegener's granulomatosis (WG) and reflects the effect of treatment. In the present enzyme-linked assay, immunoplates were coated with the cytoplasmic alpha fraction of neutrophils obtained from apparently healthy human donors by nitrogen bomb cavitation and subsequent Percoll gradient centrifugation. Alkaline phosphatase-labelled anti-human IgG was used as a secondary antibody. Diluted sera from 70 patients with WG and 16 patients with other diseases with anti-myeloperoxidase antibodies (anti-MPO) were examined. It is concluded that the ELISA accurately detects IIF ANCA positive patients with WG, is helpful in detecting WG patients in remission, is not influenced by the presence of anti-MPO and may help in detecting ANCA in cases with granulocyte-specific anti-nuclear antibodies since this IIF pattern obscures the IIF ANCA patterns. The ELISA with titration can be carried out in 3.5 h whereas a rapid test just to detect ANCA can be performed in 30 min.     

Type IV collagen alpha 5 chain. Normal distribution and abnormalities in X-linked Alport syndrome revealed by monoclonal antibody.

Although the evidence indicates that mutation of the gene for the alpha 5 chain of type IV collagen, alpha 5-(IV), is the primary defect in X-linked Alport syndrome, protein data for the alpha 5(IV) chain with regard to its normal distribution and its distribution in patients with Alport syndrome is lacking. We produced a rat monoclonal antibody (H51) by immunizing rats with a synthetic peptide corresponding to the nonconsensus amino acid sequence of alpha 5(IV) NC1 domain. H51 reacted by Western blotting with 26-kd cationic monomers and associated dimers of human type IV collagen NC1 domain. Immunohistochemical studies demonstrated that in normal human kidney alpha 5(IV) was present in the glomerular basement membrane and basement membranes of the Bowman's capsule and in some tubules (collecting ducts). The alpha 5(IV) chain was also detected in the basement membranes of normal skin, eye, and lung. Male patients with X-linked Alport syndrome revealed no reactivity of renal and epidermal basement membranes with H51, whereas alpha 5(IV) staining was normal in the glomerular basement membrane of patients with other types of glomerular diseases, including benign familial hematuria. The staining was also normal in the skin of nonaffected males in X-linked Alport families. Female heterozygous for Alport syndrome exhibited a discontinuous or mosaic pattern in the immunofluorescent staining of the epidermal basement membrane. These findings confirm that in patients with X-linked Alport syndrome there are abnormalities in alpha 5(IV) in renal and epidermal basement membranes at the protein level. Immunofluorescent staining of skin biopsies with this antibody may be of value in making a diagnosis of Alport syndrome, and, furthermore, may aid in detecting carrier females in whom urinary abnormalities are often mild or silent.     

Identification of a clinically relevant immunodominant region of collagen IV in Goodpasture disease

BACKGROUND: The characteristic feature of Goodpasture disease is the occurrence of an autoantibody response to the noncollagenous domain of the alpha3 chain of type IV collagen [alpha3(IV)NC1] in the alveolar and glomerular basement membrane. These antibodies are associated with the development of a rapidly progressive glomerulonephritis, with or without lung hemorrhage, whereas autoantibodies specific for the other alpha chains of the heterotrimeric type IV collagen probably do not cause disease. In this study, we have investigated whether differences in fine specificity of autoimmune recognition of the alpha3(IV)NC1 correlate with clinical outcome. METHODS: For mapping of antibody binding to type IV collagen, chimeric collagen constructs were generated in which parts of the alpha3(IV)NC1 domain were replaced by the corresponding sequences of homologous nonreactive alpha1(IV). The different recombinant collagen chimeras allowed the analysis of antibody specificities in 77 sera from well-documented patients. RESULTS: One construct that harbors the aminoterminal third of the alpha3(IV)NC1 was recognized by all sera, indicating that it represents the dominant target of the B-cell response in Goodpasture disease. Seventy percent of the samples recognized other parts of the molecule as well. However, only reactivity to the N-terminus of the alpha3(IV)NC1 correlated with prognosis, that is, kidney survival after six months of follow-up. CONCLUSION: The results indicate the crucial importance of antibody recognition of this particular domain for the pathogenesis of Goodpasture disease, thereby opening new avenues for the development of better diagnostic and therapeutic procedures.     

Inflammation markers in nasal lavage, and nasal symptoms in relation to relocation to a newly painted building: a longitudinal study

Introduction: There is a need to evaluate possible health effects of ventilation improvements and emissions from new buildings, in longitudinal studies. New methods to study biological effects on the eyes and upper airways are now available. Material and methods: A longitudinal study was performed on 83 trained social workers in two offices in Uppsala, Sweden. The exposed group (n= 57) moved to a newly redecorated building nearby. Low emitting building material had been used, including a new type of solvent-free water-based paint. The control group (n= 26) worked in the same office during the study period (November 1995 to February 1996). Hygiene management was carried out in both offices, at the beginning and the end of the investigation. Tear film stability (BUT) was measured. Nasal patency was measured by acoustic rhinometry, and eosinophilic cationic protein (ECP), myeloperoxidase (MPO), lysozyme and albumin were analyzed in nasal lavage fluid (NAL). Results: The relocation resulted in an increase in the personal outdoor airflow rate from 11 to 22 l/s. Indoor concentrations of terpenes were higher in the new building, and powdering of the new linoleum floor was observed. Measurements showed low levels of volatile organic compounds (VOC), formaldehyde, carbon dioxide (CO₂), nitrogen dioxide, respirable dust, and microorganisms in the air of all buildings. The move resulted in an increased nasal patency and an increase of ECP and lysozyme in NAL, after adjusting for changes in the control group. No changes were observed for nasal or ocular symptoms. A seasonal effect, with a decrease of ECP, was observed in the control group. Conclusion: A well-ventilated office building can be redecorated without any major ocular or nasal effects, or measurable increase of indoor air pollution if low-emitting building materials are selected. In agreement with previous evidence, the improved ventilation flow may explain the increase of nasal patency. The increase of ECP and lysozyme in NAL suggested an inflammatory effect in the new building. Since this building had increased ventilation flow, increased concentrations of terpenes, and powdering from the polish on the new linoleum floor, identification of causative agents was difficult. The hygiene measures did not give any evidence that emissions from the new type of solvent-free water-based paints or building dampness were responsible for the observed nasal effects. Considering the higher emissions of VOC reported from older types of water-based latex paints and solvent-based wall paints, the new type of solvent-free water-based paint seems to be a good choice from the hygiene point of view. Received: 21 December 1998 / Accepted: 20 June 1999     

Tissue expansion in the head and neck. A 6-year review

More than 100 patients (38 in the head and neck) have been treated by the insertion of tissue expanders since the technique was introduced six years ago. Our methods have been refined as we have learned more, and these improvements are described. Morbidity is high when untrained surgeons start to use the technique. The most important decision is the planning of the expander and filling port pockets, but above all the location of the incision(s): Incisions must be kept small and away from the defect, the pocket, and the future flap. Intraoperative filling of the expander reduces the need for drains by preventing haematoma and seroma formation, and reduces the formation of expander envelope folds. The optimal location of the valve is a "quiet" area above or lateral to (or both) the expander, and at least 7 cm away. Mathematical formulas are useless in predicting available flap length, as elasticity and contractility depend on individual factors. A good estimation of flap length is twice the height of the expander above the skin surface or the distance over the dome of the expander minus the corresponding measurement of its base. Overexpansion by 30-50% makes the procedure more predictable.     

The prevalence and clinical significance of alpha 1-antitrypsin deficiency (PiZ) and ANCA specificities (proteinase 3, BPI) in patients with ulcerative colitis

Our aim was to determine the prevalence of the PiZ allele for alpha 1-antitrypsin (AAT) deficiency and some relevant antineutrophil cytoplasmic antibody (ANCA) specificities in patients with ulcerative colitis (UC), and explore a possible association between these markers. In addition, we studied the relation to disease extension and activity. Sera from 141 patients with UC (72 women) were analyzed while 50 blood donors and 54 patients with acute myocardial infarction served as controls. Serum samples were screened for PiZ with ELISA and phenotyped by isoelectric focusing. BPI-ANCA and PR3-ANCA were detected by ELISA. Results were that 8.5% (12/141) of the patients with UC were PiZ carriers, higher than expected in the general Swedish population (4.7%) (p = 0.03). There was a significant difference between PiZ-carriers and non-PiZ-carriers in the extension and severity of colitis (odds ratio = 4.1, confidence interval = 1.1, 14.9; p = 0.028, and odds ratio = 9.0, confidence interval = 1.1, 73.3; p = 0.015; respectively). BPI-ANCA and PR3-ANCA were detected in 20.5% (29/141) and 12% (17/141) (p < 0.05 compared with controls for all parameters). Occurrence of BPI-ANCA and PR3-ANCA was not related to extension or severity of colitis (p > 0.05 for both variables). We observed no association between PiZ-carrier status and occurrence of BPI-ANCA or PR3-ANCA. The increased frequency of heterozygosity for the PiZ variant of AAT deficiency among patients with UC might imply a role played by protease inhibitors for regulation of inflammation and immunologic response in UC.