Effects of lactoferrin on rat dermal interstitial fluid pressure (Pif) and in vitro endothelial barrier function.

We recently demonstrated that intravenous (i.v.) injection of the iron-binding protein lactoferrin (Lf) followed by antilactoferrin (aLf) antibodies or iron-saturated Lf alone increased albumin extravasation in vivo in several tissues including skin. Increased driving pressure for blood-tissue exchange or direct effects of Lf on the endothelial barrier are possible mechanisms. We therefore, firstly, measured interstitial fluid pressure (Pif) in dermis of rats given 1 mg Lf i.v. followed 30 min later by aLf or saline and circulatory arrest 1 or 5 min thereafter and compared with controls. Secondly, transmonolayer passage of Evans blue labelled albumin (EB-albumin) was evaluated in porcine pulmonary artery endothelial cells exposed to iron-free or iron-saturated Lf (both 100 microg mL-1) in the absence and presence of 0.5 mM hydrogen peroxide. Pif increased significantly at 11-30 min following Lf to +2.1 +/- 0.3 and +1.7 +/- 0.2 mmHg at 11-20 and 21-30 min, respectively, compared with +0.1 +/- 0.2 mmHg before Lf (P < 0.05, n=25). Endothelial transmonolayer passage of EB-albumin during 3 h was not affected by iron-free or iron-saturated Lf neither in the absence nor presence of hydrogen peroxide that increased passage 3.5 times compared with controls. In conclusion, Lf-induced increase in albumin extravasation in rat skin is not explained by changes in Pif (because Lf raised Pif significantly) or direct effects of Lf on the endothelial barrier.

Transportation and storage of cryosections in PBS-Dulbecco.

Tissue pieces that cannot be fixed and embedded before immunostaining must be cryosectioned and stained quickly. We describe an easy and reliable technique to keep cryosections with immune complexes and complement stored for several weeks until the final immunostaining. The same technique was used for long distance transportation of cryosections and it was extensively tested for human kidney biopsies with excellent results.

Ki-67 antigen expression as a prognostic factor in primary and recurrent astrocytomas.

BACKGROUND AND PURPOSE: Histological grading is widely used to evaluate the prognosis for patients with astrocytic tumors. Many complimentary methods have been introduced, such as proliferation markers in order to better assess the proliferative potential of these gliomas. METHODS: Archival, paraffin embedded specimens of recurrent astrocytic tumors of varying grades from 22 patients were examined using antibodies against Ki-67 (MIB-1). Labeling indices (LI) were analyzed at the first and second operations and compared with tumor grades, age of the patients and the time between the operations as well as the survival time. RESULTS: There was a progression of malignancy between the operations. Dividing Ki-67 labeling indices in < or = 10% vs. > 10% significantly separated parameters such as the time between the first operation and relapse as well as the cumulative proportion of survival. The proliferation fraction was an independent prognostic factor. CONCLUSION: Assessment of Ki-67 LI is highly recommended in addition to histology in evaluation of the malignancy potential of astrocytic tumors.

Effect of anticoagulation upon nephron obstruction in experimental acute ischaemic renal failure. A morphological study.

Ischaemic-reperfusion injury as a model of acute renal failure (ARF) results in increased macromolecular permeability, tubular obstruction, and renal oedema. To investigate the role for coagulation in this model, anticoagulated and saline-pretreated rats were subjected to 60 min unilateral renal artery occlusion (RAO). After 15 min of reflow, specimens were collected for electron and light microscopic examination. Morphometry was employed to study podocyte changes and Bowman's space dilatation as measures of increased permeability and tubular obstruction, respectively. After 15 min of reflow, Bowman's space increased significantly and the podocytes were markedly widened and flattened. Rats pretreated with heparin or warfarin showed less widening of Bowman's space than saline-treated rats, whereas no significant difference was seen regarding the podocyte changes. In saline-treated rats, fibrin-positive material was seen in the tubules but not in the urine sediments collected after 90 min of reflow, either due to fibrinolysis or poor urinary elimination. The results suggest that anticoagulation does not preclude the glomerular sieving of macromolecules, but seems to reduce tubular obstruction, probably by preventing conversion of filtered fibrinogen into fibrin.

Allergic nephropathy associated with norfloxacin and ciprofloxacin therapy. Report of two cases and review of the literature.

Allergic nephropathy associated with quinolone antibiotics has been reported in an increasing number of cases. The mechanism might be a hypersensitivity reaction. Norfloxacin has been incriminated previously as a cause once only, with acute interstitial nephritis (AIN) as the histopathological finding. Ciprofloxacin-associated nephropathy has been reported in 28 cases, with AIN as the main histopathological finding. This report describes a second case of AIN associated with norfloxacin treatment and another ciprofloxacin-associated renal interstitial drug adverse reaction. Clinicians should be aware of quinolone-associated AIN, which is a rare but potentially dangerous renal complication.

Prolactin-derived amyloid in the aging pituitary gland.

Small amyloid deposits occur commonly in different organs in association with aging. As in other amyloids, the fibrils in the age-associated forms are built up by specific proteins, unique to every histological type. The amyloid proteins that have been identified in localized amyloid of human endocrine organs have all been of polypeptide hormone nature, and these include calcitonin, islet amyloid polypeptide (amylin), and atrial natriuretic factor. In the present study, we add prolactin to the increasing group of known amyloid proteins and show that this hormone constitutes the amyloid fibrils of pituitary glands of aging individuals.

A study of autoantibodies and circulating immune complexes in mercury-exposed chloralkali workers.

UNLABELLED: Inorganic mercury may cause immunologically mediated disease: e.g., glomerulonephritis, acrodynia, and contact allergy. Animal models have demonstrated the importance of genetic factors in determining susceptibility and resistance to autoimmunity, as well as the specific manifestation of the autoimmune response. Findings in groups of workers with occupational exposure to inorganic mercury have been inconsistent. OBJECTIVE: To investigate whether an immune response, caused by exposure to inorganic mercury (Hg), could be shown in occupationally exposed workers. METHODS: Immunoglobulin G (IgG), antinuclear autoantibodies, antibodies against thyroid, stomach or kidney antigens using indirect immunofluorescence, antibodies against glomerular basement membrane using ELISA, and circulating immune complexes in serum, and albumin in urine, were examined in Hg-exposed workers and controls. The two groups, 41 male chloralkali workers exposed to Hg vapour (mean exposure time 9 years) and 41 unexposed controls were age-matched and recruited from the same company. Hg concentrations in whole blood (B-Hg), plasma (P-Hg), and urine (U-Hg) were determined using cold vapor atomic spectrometry. DESIGN: Cross-sectional study. RESULTS: The mean B-Hg, P-Hg and U-Hg levels were 46 nmol/l, 37 nmol/l, and 27 micrograms/g creatinine in the exposed group, and 17 nmol/l, 6.9 nmol/l, and 3.4 micrograms/g creatinine in the referents. No statistically significant differences were found regarding IgG levels, urinary albumin excretion, prevalence of abnormal titers of autoantibodies or circulating immune complexes. There were no statistically significant associations between autoantibodies or immune complexes on the one hand and mercury exposure indices on the other. CONCLUSION: The results indicate that, if and when lasting autoimmune response occurs at the mercury exposure levels of the present study, it is uncommon. A small fraction of humans may, however, be susceptible to the development of autoimmunity, and there is also a possible "healthy worker" selection. Thus cross-sectional studies of moderate numbers of active workers will have low power to demonstrate autoimmune effects.

Dermal IgG deposits and increase of mast cells in patients with fibromyalgia--relevant findings or epiphenomena?

Skin biopsies from 25 patients with fibromyalgia, 5 healthy controls, 8 patients with rheumatoid arthritis, and 9 patients with local chronic pain after whiplash injury, were examined for the occurrence of IgG deposits and collagen types, using direct and indirect immunofluorescence, and for dermal connective tissue mast cells, using semithin Epon sections. Fibromyalgia skin biopsies had significantly higher values of IgG deposits in the dermis and vessel walls and showed a higher reactivity for collagen III. They also had a higher mean number of mast cells. There was a correlation between the percentage of damaged/degranulated mast cells and the individual IgG immunofluorescence scores. These findings support the hypothesis of neurogenic inflammation involvement in fibromyalgia.

DNA ploidy and S-phase in recurrent astrocytomas: a retrospective study by flow cytometry of deparaffinized specimens.

Twenty-two patients with recurrent astrocytic tumors were treated surgically two or even three times. At the time of the first surgery 6 tumors were fibrillary astrocytomas (grade II), 9 anaplastic astrocytomas (grade III) and 7 glioblastomas (grade IV). Histopathological specimens from second surgery demonstrated in 12 cases signs of higher grades of malignancy. Flow cytometry (FCM) did not reveal any significant changes of S-phase fraction (p = 0.55). This study supports the theory that, given enough time, the histopathology of brain tumors change significantly from more benign forms to more malignant ones. The flow cytometry (FCM) could trace a weak tendency to higher S-phase fractions at the time of the second surgery. No apparent change of ploidy pattern was observed. In spite of the unequivocal histopathological changes of the recurrent astrocytomas the flow cytometry failed to indicate similar changes in terms of ploidy and S-phase fraction parameters.

Prognostic value of flow cytometry and correlation to some conventional prognostic factors: a retrospective study of archival specimens of 134 astrocytomas.

The S-phase fraction and the DNA ploidy type in 134 astrocytomas (18 Grade II, 46 Grade III, and 70 Grade IV astrocytomas) were studied using flow cytometry in a retrospective study of archival tumor specimens. A high grade of malignancy was associated with both a high S-phase fraction (p < 0.0001) and an aneuploid DNA pattern (p < 0.0001). There was no aneuploid DNA pattern found in the fibrillary astrocytomas (World Health Organization (WHO) Grade II); where-as the aneuploid pattern was observed in 80% of all the glioblastomas multiforme (WHO Grade IV). The age and gender of the patients were not significantly related to the flow cytometry parameters. The survival of patients with Grade II or III astrocytomas was significantly longer when their tumors exhibited a tetraploid DNA pattern or had a low S-phase fraction. In patients with Grade IV tumors, there was no correlation between length of survival and either the DNA ploidy or the S-phase fraction. In a multivariate Cox regression analysis of data obtained in patients with Grade II and III astrocytomas, age, grade of malignancy, DNA ploidy, and S-phase fraction were independent prognostic factors.

Fat cylinder transplantation: an experimental comparative study of three different kinds of fat transplants.

In an experimental comparative study, fat cylinders harvested with a new instrument were compared with excised fat and aspirated fat. In 12 New Zealand White rabbits, fat grafts of about 1 ml were transplanted from the fat pad between the shoulders to the scalp and rear side of the ears by three different fat harvesting techniques. After 6 months, the change in the weight of each of the 36 specimens was measured. All specimens were freeze-cut after fixation and stained with Sudan IV, a fat-specific stain. They were examined under a light microscope and evaluated by computer-assisted image analysis. There was no statistical difference in the percentage change in weight between the excised fat and the fat cylinder groups (2 and 1 percent, respectively). For aspirated fat, however, the difference was significant (-59 percent). There also were significantly more surviving mature adipocytes in the fat cylinder group than in the aspirated fat group. We conclude that fat cylinders harvested with the new instrument are as good grafting material as excised fat, while aspirated fat in this study was clearly inferior for grafting.

Urolithiasis and distal renal tubular acidosis preceding primary Sjögren's syndrome: a retrospective study 5-53 years after the presentation of urolithiasis.

OBJECTIVES: Distal renal tubular acidosis (dRTA) can be associated with autoimmune diseases such as primary Sjögren's syndrome (SS). Our objective was to study SS-associated symptoms, autoantibodies and renal histopathology in patients with urolithiasis and dRTA. SETTING: The patients were from the Departments of Nephrology and Rheumatology. University Hospital of Linköping, which is a tertiary referral hospital, as well as a secondary referral centre for the immediate area around the city of Linköping. SUBJECTS: Ten female patients with dRTA, who presented with urolithiasis and not with subjective sicca symptoms, were from the Department of Nephrology, University Hospital, Linköping. Autoantibodies were detected in eight of these patients, and they were studied with respect to clinical and laboratory evidence of SS (urolithiasis group). Fifteen women with SS, who presented with sicca symptoms and not with urolithiasis or dRTA, served as the reference group. RESULTS: In the urolithiasis group, all of the eight patients had anti-SS-A antibodies, and SS (or possible SS) developed in seven of the eight patients 1-48 (mean 15) years after the onset of urolithiasis. Histological features of tubulointerstitial nephritis were found in four of five biopsied patients in the urolithiasis group, and in two of four patients (with dRTA) in the reference group. CONCLUSIONS: Urolithiasis and dRTA can precede subjective sicca symptoms, and patients with dRTA may have SS in the absence of subjective sicca symptoms. Anti-SS-A antibodies are common in patients with urolithiasis and dRTA. Therefore, we hypothesize the possibility of a Sjögren-related renal disease in these patients.

Murine genotype influences the specificity, magnitude and persistence of murine mercury-induced autoimmunity.

Genetic factors are major contributors in determining the susceptibility to systemic autoimmune diseases. We studied the influence of genotype on systemic autoimmunity by treating female mice of the H-2s strains SJL/N, SJL/J, A.SW, and B10.S with mercuric chloride (HgCl2) for 10 weeks and then following autoantibody and tissue immune deposits during the subsequent 12 months. All strains developed antinucleolar antibodies (ANoA) of the IgG class which reacted in immunoblotting with a 34-kDa nucleolar protein identified as fibrillarin. The titre of ANoA attained after 10 weeks' treatment varied from 1:1,280 to 1:20,480 in the order: A.SW > SJL > > B10.S. Following cessation of HgCl2 treatment ANoA and antifibrillarin antibodies (AFA) persisted for up to 12 months, although some B10.S mice showed pronounced reduction not only of their autoantibody titres, but also systemic immune deposits when compared to other H-2s strains. A second set of autoantibodies targeted chromatin and in some mice specifically histones, and were distinguished from the ANoA by a rapid decline after treatment and a susceptibility linked to the non-H-2 genes of the SJL. Tissue levels of mercury remained elevated above untreated controls throughout the study period, suggesting that the mercury detected in lymphoid tissues may provide stimulation of lymphoid cells specific for fibrillarin for a considerable period after exposure has ceased. We conclude that H-2 as well as non-H-2 genetic factors distinctly influence not only the susceptibility to induction of autoimmunity, but also the specificity and magnitude of the response.

Distribution of lactoferrin and 60/65 kDa heat shock protein in normal and inflamed human intestine and liver.

Immunisation against the mycobacterial heat shock protein (hsp-65) has been proposed to lead to production of autoantibodies against human lactoferrin. Such antibodies occur in ulcerative colitis and in primary sclerosing cholangitis. This study analysed the distribution of hsp-65 and lactoferrin in biopsy specimens from patients with inflammatory bowel disease and primary sclerosing cholangitis and studied whether immunisation against mycobacterial hsp-65 resulted in production of antilactoferrin antibodies and vice versa. Polyclonal rabbit antihuman lactoferrin and monoclonal mouse anti-hsp-65 (ML30) were used for immunohistochemistry on biopsy specimens from patients with inflammatory bowel disease and primary sclerosing cholangitis. Rats were immunised against human lactoferrin and mycobacterial hsp-65 respectively. Antibody measurements were done by enzyme immunosorbent assays. It was found that lactoferrin and hsp-60/65 were not codistributed. Lactoferrin was found on vascular endothelium and in nonparenchymal liver cells both in inflamed and uninflamed tissues, but only in the hepatocytes of inflamed liver. ML30 reactivity was not inhibited by antilactoferrin antibodies. Rat anti-hsp-65 serum had no detectable antilactoferrin antibodies. In conclusion, antilactoferrin antibodies probably do not arise by immunisation against mycobacterial hsp-65. Both nonparenchymal cells and hepatocytes probably participate in clearance of lactoferrin. Endothelial exposure of lactoferrin may have pathogenic implications in diseases with antilactoferrin autoantibodies.

Histopathology of renal biopsies with correlation to clinical findings in primary Sjögren's syndrome.

OBJECTIVE: To characterise the renal histopathological lesions in patients with primary Sjögren's syndrome and to correlate the findings with the clinical manifestations. METHODS: Kidney and labial salivary gland biopsies from ten patients with primary Sjögren's syndrome were studied. The protocol included semiquantitative assessments of the inflammatory focus scores and the levels of atrophy, interstitial fibrosis, glomerular sclerosis and vessel wall thickening. RESULTS: No statistically significant correlation was found between the kidney and labial inflammatory scores; this was probably the result of observation of the lesions at different stages. The level of chronic tubulointerstitial nephritis was correlated to the glomerular filtration rate and to the extent of glomerular sclerosis but not to vessel wall thickening. These findings indicate a primary injury of the tubules in primary Sjögren's syndrome that secondarily compromises the glomeruli. CONCLUSION: A semiquantitative approach to the assessment of histopathological features, as used in this study, appears to be valuable for characterising the lesions in kidney biopsies.

Resin embedding for quantitative immunoelectron microscopy. A comparative computerized image analysis.

Quantitative immunoelectron microscopy (QIEM) is dependent on the reliability of preparative techniques for both efficient immunolabeling and consistent quantitative results among series of immunostained sections. The present study compared Lowicryl K4M and Epon embedding after identical fixation and dehydration of rat somatotrophic secretory granules. Labeling intensity, diameter, roundness, uptake of uranyl acetate, and gray value were measured with computer assisted image analysis. Lowicryl-embedded granules showed the highest labeling densities after conventional fixation and Progressively Lowering Temperature (PLT) dehydration, but values were not consistent in a series of immunostained sections. A lower but more uniform level of immunostaining was seen in Epon-embedded sections when tissue was cryofixed and physically dehydrated. Gray value measurements from micrographs from both embedding media confirmed the better contrast of Epon sections and the different reliefs of the granule surfaces. This study emphasizes the importance of complete comparisons of preparative techniques for QIEM for reliability and reproducibility.

Does amalgam affect the immune system? A controversial issue.

Although in use for more than 150 years, dental amalgam has been questioned more or less vigorously as a dental restoration material due to its alleged health hazard. Humans are exposed to mercury and the other main dental amalgam metals (Ag, Sn, Cu, Zn) via vapour, corrosion products in swallowed saliva, and direct absorption into the blood from the oral cavity. Dental amalgam fillings are the most important source of mercury exposure in the general population. Local, and in some instances, systemic hypersensitivity reactions to dental amalgam metals, especially mercury, occur at a low frequency among amalgam bearers. Experimental and clinical data strongly indicate that these and other subclinical systemic adverse immunological reactions to dental amalgam metals in humans will be linked to certain MHC genotypes, and affect only a small number of the exposed individuals. These individuals will be very difficult to detect in a mixed population of susceptible and resistant individuals, including persons with alleged symptoms due to dental amalgam fillings, where many of the individuals are likely to suffer from conditions with no proven immunological background such as multiple chemical sensitivity syndrome. Intensified studies should be performed to identify such susceptible MHC genotypes, taking advantage of the reported cases of more heavily metal-exposed humans with systemic autoimmune reactions. Further studies will also be needed to ascertain whether the combined exposure to the metals in dental amalgam may lower the threshold for adverse immunological reactions, since recent studies have shown that the metals in alloy, especially silver, may induce autoimmunity in genetically susceptible mice.

Adverse immunological effects and autoimmunity induced by dental amalgam and alloy in mice.

Dental amalgam fillings are the most important source of mercury exposure in the general population, but their potential to cause systemic health consequences is disputed. In this study, inbred mice genetically susceptible to mercury-induced immune aberrations were used to examine whether dental amalgam may interfere with the immune system and cause autoimmunity. Female SJL/N mice were implanted in the peritoneal cavity with 8-100 mg silver amalgam or silver alloy for 10 weeks or 6 months. Chronic hyperimmunoglobulinemia, serum IgG autoantibodies targeting the nucleolar protein fibrillarin, and systemic immune-complex deposits developed in a time- and dose-dependent manner after implantation of amalgam or alloy. Splenocytes from mice implanted with amalgam or alloy showed an increased expression of class II molecules. The functional capacity of splenic T and B cells was affected in a dose-dependent way: 10 weeks of low-dose and 6 months of high-dose amalgam implantation strongly increased mitogen-induced T and B cell proliferation, whereas 10 weeks of high-dose implantation decreased the proliferation. Not only mercury but also silver accumulated in the spleen and kidneys after amalgam implantation. In conclusion, dental amalgam implantation in a physiological body milieu causes chronic stimulation of the immune system with induction of systemic autoimmunity in genetically sensitive mice. Implantation of silver alloy not containing mercury also induced autoimmunity, suggesting that other elements, especially silver, have the potential to induce autoimmunity in genetically susceptible vertebrates. Accumulation of heavy metals, from dental amalgam and other sources, may lower the threshold of an individual metal to elicit immunological aberrations. We hypothesize that under appropriate conditions of genetic susceptibility and adequate body burden, heavy metal exposure from dental amalgam may contribute to immunological aberrations, which could lead to overt autoimmunity.

Selective induction of anti-fibrillarin autoantibodies by silver nitrate in mice.

Female SJL (H-2s) mice developed serum IgG anti-nucleolar antibodies (ANoA) after 5 weeks treatment with 0.05% or 0.01% silver nitrate (AgNO3) in drinking water. Five more weeks of treatment increased the ANoA titre to 3410 +/- 853 and 640 +/- 175 (reciprocal mean +/- s.e.m.), respectively. Controls receiving ordinary tap water and mice given 0.002% AgNO3 showed no antinucleolar antibodies. The high-titre ANoA targeted a 34-kD nucleolar protein identified as fibrillarin, the major autoantigen in murine mercury-induced autoimmunity and in a fraction of patients with systemic scleroderma. Serum autoantibodies to chromatin or histones, kidney, spleen, stomach, thyroid, or skin antigens (except the nucleolus) were not found in any of the mice. There was no consistent significant increase of serum IgG1, IgG2a, IgG2b, or IgG3 concentrations after AgNO3 treatment compared with controls. Mice treated with 0.05% AgNO3 for 10 weeks showed a slight decrease in serum IgG1, IgG2b and IgG3 concentrations. These mice also showed a small but statistically significant increase in renal, mesangial IgM deposits, which was not accompanied by any increase in C3c deposits, whereas mice given lower doses of silver nitrate showed no significant increase in mesangial immunoglobulin immune deposits. Systemic vessel wall immune deposits were not found in any of the mice. In mice given 0.05% silver nitrate, the kidney showed the highest concentration of silver (12.2 +/- 0.09 micrograms Ag/g wet weight; mean +/- s.e.m.), followed by the spleen (8.7 +/- 1.3), and the liver (3.9 +/- 0.4). Treatment with 0.01% silver nitrate caused a different distribution of silver, with the highest concentration in the spleen (2.1 +/- 0.16 micrograms Ag/g), followed by the kidney (0.63 +/- 0.037), and the liver (< 0.29 micrograms Ag/g; mean). Silver seems to be a more specific inducer of antinucleolar/anti-fibrillarin autoantibodies than mercury and gold, lacks the general immune stimulating potential of mercury, and has only a weak tendency to induce renal immune deposits. These observations suggest that the autoimmune sequelae induced in mice by metals is dependent, not only upon the genetic haplotype of the murine strain, but also on the metal under investigation.

Cryofixation combined with physical dehydration for quantitative immunoelectron cytochemistry.

Common methods for preparing samples for immunoelectron microscopy involve glutaraldehyde fixation (GA) followed by chemical dehydration (CD) or cryofixation (CF) succeeded by physical dehydration, i.e., freeze drying (FD) or freeze substitution (FS). The effects of these techniques have been evaluated with regard to the sizes of epoxy resin embedded rat somatotrophic secretory granules as well as the immunolabeling densities over these granules. The measurements were performed by computerized image analysis using electron microscopy in transmission (TEM) and scanning transmission (STEM) modes, which allowed us to define the immunolabeling in detail. The embedded secretory granules showed the same diameters after GA (2 hr) with CD and GA (15 min) with CF and FS, but were smaller after CF-FS, and smallest after GA (15 min) with CF and FD. The highest labeling density appeared after GA (15 min) and physical dehydration, in particular after freeze substitution. Based on our STEM pictures a new factor for evaluating and interpreting immunolabeling of granules is introduced; the "accessible immunogold labeling surface." It defines the fraction of the epoxy resin surface that is labeled and varies with the preparation methods. By using this factor, an order of labeling densities/micron 2 over the accessible areas could be established for the different techniques: GA-CF-FS > CF-FS > GA-CF-FD > GA-CD. The high labeling after GA-CF-FS may be due to the combination of a large accessible area and accurate preservation of the antigenicity of the hormones in the granules.