Application of ELISA for IgM, IgA and antigen detection for rapid diagnosis of respiratory syncytial virus infections: a comparative study.

BACKGROUND: Infection with Respiratory Syncytial Virus (RSV) can be rapidly diagnosed by detection of viral antigen in nasopharyngeal secretions (NPS) or serologically by detecting IgM and IgA antibodies. OBJECTIVES: To evaluate the above methods for reliability and rapidity, and compare them with the complement fixation (CF) test and virus isolation. STUDY DESIGN: Viral antigen was tested in 145 NPS samples by ELISA in parallel with tissue culture isolation. The geometric mean titer (GMT) of complement fixing antibodies in various age groups was determined from 92 individual serum samples by CF test. The diagnostic methods, CF test, ELISA-IgM and ELISA-IgA, were evaluated using 21 pairs of acute and convalescent sera. Appearance of IgM and IgA in serum samples with low or negative CF titers was studied in two age groups: 0-10 months (n = 82), and 11 months-9 years (n = 47). RESULTS: From the 145 NPS samples, 20 samples were positive by both ELISA and virus isolation, 9 were positive only by ELISA and 5 were positive only by virus isolation. The GMT by age group for 92 sera evaluated by CF test were 40 (0-10 months), 195 (11-24 months), 269 (2-4 years), 173 (5-12 years) and 132 (adults). Among the 21 paired sera examined, CF test detected 13 RSV infections by antibody rise or seroconversion while the ELISA-IgM/IgA tests identified all 21 infections, 7 of them in the first sample. The presence of IgM alone or IgM and IgA antibodies was demonstrated in both age groups examined; however, IgA alone was found only in the age group 11 months and older. CONCLUSIONS: ELISA for antigen detection is better than virus isolation because it is faster (6-20 h vs. 3-20 days, respectively) and more sensitive (29/34 vs. 25/34 positives, respectively). ELISA-IgM and ELISA-IgA was more sensitive and reliable than the CF test, particularly for the 0-10 month age group. Thus, when necessary, serological diagnosis of RSV infection can be based on the presence of IgM and/or IgA antibodies in serum samples obtained early after onset of symptoms.

Uninephrectomy aggravates tubulointerstitial injury in rats with adriamycin nephrosis.

The effects of uninephrectomy on the function and structure of the remnant kidney were assessed in rats with Adriamycin-induced nephrosis, 12 weeks after the injection of Adriamycin. The kidney volume of Adriamycin-treated uninephrectomized rats (NX-AD) was 2.3 times that of sham-operated, Adriamycin-treated animals (SH-AD; p < 0.001). The marked renal enlargement in NX-AD animals was due to the development of large tubular cysts. Following uninephrectomy, the fractional volume of tubular lumen almost doubled (NX-AD, 0.33 +/- 0.02; SH-AD, 0.17 +/- 0.02; p < 0.001) and the absolute volume of tubular lumen increased more than fourfold (NX-AD, 0.51 +/- 0.08 ml; SH-AD, 0.12 +/- 0.02 ml; p < 0.001). The frequency of tubular lumen with a large cross-sectional area (> or = 40,000 microns 2) was 5.8 +/- 1.1% in NX-AD and 0.7 +/- 0.2% in SH-AD groups (p < 0.001). The fractional volume of interstitial fibrosis in NX-AD animals was larger than in SH-AD (0.09 +/- 0.02 versus 0.04 +/- 0.01%, p < 0.05). As opposed to the worsening of tubulointerstitial disease, single-kidney glomerular filtration rate, fractional protein clearance, glomerular volume and the extent of glomerular sclerosis did not differ significantly in NX-AD as compared to SH-AD groups. This study shows that uninephrectomy in rats with Adriamycin nephrosis worsens interstitial nephrosis and aggravates the formation of tubular cysts, leading to a macrocystic kidney disease. These changes are not associated with an increase in glomerular sclerosis.

Gaucher's disease and mesangiocapillary glomerulonephritis in childhood--a coincidence?

A 6-year-old boy, presenting with a nephritic syndrome, was diagnosed as suffering from Gaucher's disease (GD) and mesangiocapillary glomerulonephritis (MCGN). GD was suspected because of aseptic necrosis of the femoral heads on X-ray and later confirmed by bone marrow aspiration and a lack of glucocerebrosidase activity in white blood cells; MCGN was documented on renal biopsy. The child was treated with prednisone, dipyridamole and aspirin, and recovered completely clinically. A second biopsy was not performed. The connection between these two rare diseases, and between nephritis and GD in general, is discussed.

Increased leukocyte adhesiveness/aggregation and tissue leukostasis following surgical trauma.

The purpose of the present study was to document the increased leukocyte adhesiveness/aggregation (LAA) and tissue leukostasis that follow surgical trauma. We found that following major abdominal surgery, the percentage of aggregated leukocytes in the peripheral blood increased from 8.6 +/- 7 to 21.7 +/- 11.5 (p < 0.001), while the respective values for patients undergoing minor surgery were 3.7 +/- 2.4 and 19.7 +/- 7.6% (p < 0.001). The state of LAA was also determined in eight dogs after partial collectomy and four controls. A clear increment in the degree of tissue leukostasis (especially in the spleen, liver and lungs) was noted in the operated as compared to the control animals. In addition, a significant correlation was found between the state of LAA, determined in the venous (p = 0.0004) and arterial (p = 0.002) blood of these animals and the degree of tissue leukostasis. We conclude that a state of increased LAA in the peripheral blood is induced by surgical trauma and that this increased LAA correlates with tissue leukostasis.

The effect of cyclosporin A on early and late stages of experimental lupus.

OBJECTIVE: To investigate the effect of cyclosporin A (CSA) on the development of lupus in an experimental model. METHODS: Lupus was induced in naive mice following injection of a human anti-double-stranded DNA (anti-dsDNA) monoclonal antibody carrying the 16/6 idiotype (Id). CSA was injected into the mice at an early stage of the disease (2 months after immunization) and at a late stage (4 months after immunization). RESULTS: CSA was found to have a suppressive effect on autoantibody production, as well as on the appearance of other disease manifestations, in the mice with lupus. The effects of the drug were more prominent when the mice were treated at an early stage. This was reflected by a dramatic decrease, to normal levels, in autoantibodies to dsDNA, histones, cardiolipin, Sm, RNP, SS-A/Ro, SS-B/La, and anti-DNA 16/6 Id. Similar effects on the erythrocyte sedimentation rate, white blood cell count, and urinary protein levels were noted. These data were supported by electron microscopy analysis showing a lack of immunoglobulin deposition in the kidneys of mice in which treatment was started early. CONCLUSION: This study demonstrates that, similar to findings in other autoimmune conditions (e.g., insulin-dependent diabetes mellitus), administration of CSA at an early stage in systemic lupus erythematosus may be more beneficial than if the drug is given at a later stage.

Induction of experimental anti-phospholipid syndrome associated with SLE following immunization with human monoclonal pathogenic anti-DNA idiotype.

MIV-7 is a human monoclonal antibody that binds to DNA and carries a pathogenic anti-DNA idiotype 16/6. The antibody was generated by fusing peripheral blood lymphocytes of a healthy donor which were stimulated with an anti-idiotypic antibody to B11 (a human mAb anti-mouse mammary tumor virus-MMTV). The MIV-7, in addition to being an anti-DNA antibody, also binds to MMTV glycoproteins. Following immunization into the footpad of naive BALB/c mice with MIV-7, the mice developed anti-phospholipid syndrome (APLS) and SLE. The APLS was characterized by thrombocytopenia, the presence of anticardiolipin antibodies, lupus anticoagulant (prolonged APTT), high resorption rate of fetuses and lower mean weights of the placentae and fetuses. The SLE was characterized by serological markers (e.g. anti-DNA), laboratory (increased sedimentation rate and proteinuria) and histological findings (deposition of immune complexes in the glomeruli). Active immunization of mice with mouse monoclonal anti-cardiolipin antibodies led to the induction of primary APLS without SLE. The results add to our previous passive transfer model in which mouse monoclonal anti-cardiolipin antibody generated from immunized mice (CAM) was infused into the tail vein and also resulted in induction of pure APLS [11]. Our results demonstrate the ability to induce secondary APLS to SLE following immunization with a pathogenic idiotype of anti-DNA antibodies and to induce primary APLS with anti-cardiolipin mAb. The existence of these experimental models may permit controlled studies of novel therapeutic models.

Unusual renal deposit in mixed connective tissue disease.

We describe a patient with clinical signs of mixed connective tissue disease who developed nephrotic syndrome. The kidney biopsy revealed glomerular and vascular deposits that stained positive with Congo red and showed a green birefringence. The Congo red positivity became negative in sections treated with potassium permanganate. Peroxidase-antiperoxidase staining with anti-AA antibodies was strongly positive. Ultrastructurally, although the deposits were similar to amyloid, they were about twice the size of amyloid fibrils. To our knowledge, the deposition of an amyloidlike material that shows the histochemical and immunohistochemical features of amyloid AA but is lacking the distinctive ultrastructural characteristics of amyloid fibrils has not been described.

The state of leucocyte adhesiveness/aggregation (LAA) in the peripheral blood of burned mice: an early and sensitive inflammatory indicator and a marker of pulmonary leukostasis.

The inflammatory response during thermal injury increases the adhesiveness of white blood cells. A direct slide test was used to compare the state of leucocyte adhesiveness/aggregation (LAA) in the peripheral blood of mice subjected to a thermal injury with the findings in control animals. The state of LAA in the peripheral blood increased from baseline values of 1.1 +/- 1.1 per cent to 6.5 +/- 1.3 per cent within 1 h and to 11.0 +/- 1.2 per cent and 14.8 +/- 4 per cent after 3 and 6 h respectively following thermal injury. The respective leucocyte counts were 3075 +/- 277/mm3 (baseline), 3871 +/- 359, 3840 +/- 687 and 6395 +/- 1152 cells/mm3. The LAA values had subsided by 5 days following burning and correlated with the degree of pulmonary leukostasis. Our study suggests that the LAA is an early and sensitive marker of inflammation and that it can be used as a marker for the presence of pulmonary leukostasis during thermal injury.

Modulation of SLE induction in naive mice by specific T cells with suppressor activity to pathogenic anti-DNA idiotype.

T cells (CD8+) with specific suppressor activity against anti-dsDNA antibody (16/6 Id+) were generated in vitro. The cells were established from BALB/c-enriched T cells exposed in vitro to silica beads coated with the pathogenic anti-DNA idiotype, 16/6. The idiotype specificity of the suppressor cells was demonstrated by (a) specific induction of a decrease in proliferative response of T helper cell lines specific for the pathogenic idiotype (16/6 Id), when exposed to the idiotype, with no effect on T cell lines with other specificities, e.g., against human IgM or synthetic polypeptide. (b) Effectively suppressing in vitro antibody production of anti-16/6 antibody, employing 16/6-primed B cells and specific helper T cell line. The 16/6 Id-specific Ts cells were found to be MHC restricted. Weekly intravenous injections of 10(7) 16/6 Id-specific Ts cells given to BALB/c mice at different stages of experimental SLE disease prevented the clinical, serological, and pathological manifestations. This effect was characterized by decreased titers of autoantibodies (e.g., anti-DNA, anti-Sm antibodies) in the sera, by abolishment of the proteinuria, leukopenia, and the increased ESR, followed by decreased immunoglobulin deposition in the kidneys. Treating the mice with control IgM-specific T cells did not affect the above parameters. These studies demonstrate the ability to generate Ts cells specific for pathogenic idiotypes. The method might be employed therapeutically to modulate the course of autoimmune conditions.

Idiotype specific T-cell lines inducing experimental systemic lupus erythematosus in mice.

Immunization of mice with either antibodies bearing the 16/6 idiotype (16/6 Id) or anti-idiotypic antibodies against the 16/6 Id induces experimental systemic lupus erythematosus (SLE). We report here the establishment and characterization of 16/6 Id-specific T-cell lines from C3H.SW (H-2b) and BALB/c (H-2d) mice. Both lines proliferate specifically in response to the 16/6 Id in an H-2-restricted manner. The injection of 16/6 Id-specific T cells into syngeneic mice led to the development of experimental SLE. Furthermore, inoculation of the 16/6 Id-specific T-cell line derived from C3H.SW mice into the H-2 compatible C57BL/6 mice, which are non-responders to the 16/6 Id, induced experimental SLE. This report provides direct evidence for the role of idiotype-specific T cells in the induction of experimental SLE.

Primary malignant melanoma in the parotid gland.

Reports of primary malignant melanoma arising from the parotid salivary gland are extremely rare and, to date, have been sporadic. We report a pertinent case, and tabulate and correlate the clinical findings of the 13 cases reported thus far in the literature. The most common symptom is a progressively enlarging, asymptomatic, firm, and fixed mass. Total excision has been the established treatment of choice. The contribution of radiotherapy, chemotherapy, and immunotherapy remains unclear, and it is not possible at present to predict the outcome of treatment in individual patients. Although rare, primary malignant melanoma should be considered in the differential diagnosis of parotid tumors. The clinical significance of establishing the diagnosis of primary malignant melanoma of the parotid gland is emphasized.

Castration inhibits glomerular hypertrophy and proteinuria in uninephrectomized male rats.

Renal mass reduction may lead to glomerular hypertrophy, proteinuria and focal glomerulosclerosis (FGS) in humans and rats. In humans and rats, females are less susceptible than males to these phenomena. This study was undertaken to evaluate the effect of male rat castration on the pathogenesis of proteinuria and FGS. Urinary protein was measured in 60-day-old male and female rats. Uninephrectomy was performed in all rats, and castration in half of the males. After 180 days, proteinuria, glomerular filtration rate (GFR) and blood biochemistry were determined. Kidneys were resected, weighed and subjected to morphologic studies. Following uninephrectomy, male rats developed severe proteinuria: 132.3 +/- 40.9 mg 24 h-1, most of which was accounted for by an albuminuria of 70.9 +/- 19.3 mg 24 h-1. In contrast, protein excretion in female and castrated male rats remained within normal limits: 8.0 +/- 1.8 and 4.2 +/- 0.5 mg 24 h-1, respectively. Mean glomerular volume in male rats was 1.18 +/- 0.08 x 10(6) microns3; much higher than in female rats, 0.84 +/- 0.04 x 10(6) micron3, and castrated male rats, 0.87 +/- 0.03 x 10(6) micron3 (P less than 0.005). On light and electron microscopy, glomeruli of female and castrated male rats were completely normal. In contrast, in four of seven male rats, mild glomerular changes were observed. They consisted mainly of mesangial expansion, electron-dense deposits and collapse of capillary loops. These data suggest that castration confers protection against the development of glomerular hypertrophy and proteinuria in uninephrectomized male rats. Endogenous testosterone may be associated with this development.

Use of modified fine needle aspiration for study of glomerular pathology in human kidneys.

In routine fine needle aspiration (FNA) of the kidney, the glomeruli are seldom visualized. They appear as multi-layered, cellular conglomerates and, therefore, are unsuitable for morphological analysis. A novel plasma-clot technique for collection of glomeruli from FNA samples was used in a study of 6 native and 24 transplanted human kidneys with suspected glomerular lesions. This technique produced a satisfactory yield of well preserved glomeruli and enabled the identification of glomerular pathology with the accuracy comparable to that of renal core biopsy. The FNA plasma clot method may prove useful in the study of glomerular pathology under conditions where the use of percutaneous biopsy is conventionally limited or avoided.

The state of leukocyte adhesiveness/aggregation in the peripheral blood of patients with respiratory tract infections.

The state of leukocyte adhesiveness/aggregation (LAA) in the peripheral blood has been employed as a marker of inflammation. In the present study we examined patients with varying intensities of inflammation caused by respiratory tract infections to further investigate the reliability of the state of LAA for the detection and assessment of the severity of disease activity. The study includes 140 controls, 46 patients with upper respiratory tract infection, 30 with bronchitis, 27 with suspicion of pulmonary infiltrate, and 39 with small and 18 with large pulmonary infiltrate. Assessment was based on assuming an increasing severity of inflammation from the 1st to the 6th diagnostic category and by making use of discriminant analysis. It was found that the state of LAA proved to be the best variable to classify the patients into their diagnostic category (F to enter 27), followed by erythrocyte sedimentation rate at the 1st h (F to enter 20.8) and total white blood cell count (F to enter 8.3). These studies were followed by animal experimentation. A highly significant correlation (p = 0.005) was found between the state of LAA in the peripheral blood and the degree of pulmonary leukostasis in a model of endotoxemia in rabbits. These results suggest that the state of LAA is not an epiphenomenon and represents the tendency of the white blood cells to stick to the endothelium which facilitates their migration into the tissues.

Sarcoidosis with oral involvement.

A case of sarcoidosis with bilateral hilar lymphadenopathy and multiple submucosal papular oral mucosa lesions is presented. The clinical signs and symptoms described in this case are not uncommon, and their distinguishing features may expedite accurate diagnosis. Early incisional biopsy of the oral mucosa appears useful for histopathologic proof of the diagnosis.

Ultrastructural abnormalities in endoscopically and histologically normal and involved colon in ulcerative colitis.

Twenty colonoscopies (eight complete or almost complete; 12 short) were carried out on 15 patients with ulcerative colitis with the aim of comparing the endoscopic aspects with the light and electron microscopic features in biopsies taken from multiple sites. Patients with severe attacks were examined without prior preparation (two examinations). When the attack was mild to moderate (11 examinations), or the patient was in remission (seven examinations), two saline enemas were given up to 1 h before examination. There was a favorable correlation between the endoscopic and light microscopic features in 94.7% of the biopsies (total number of biopsies, 76). The electron microscope findings greatly exceeded those observed by light microscope and indicated that the major abnormality resides within the colonic epithelial cells. Distinctive ultrastructural changes were present both in apparently uninvolved (endoscopically and histologically) parts of colon and in inactive stages of ulcerative colitis. These findings suggest that colonic mucosal involvement may be universal, persist during clinical remission, and precede the light microscopic findings. They also support the importance of maintenance therapy.

Mechanical, biochemical, and structural effects of vitamin D deficiency on the chick heart.

The effects of vitamin D deprivation on the chick heart were investigated from three aspects: cardiac contractility (+/- dP/dT), intracellular high-energy phosphorus compounds, and structural differences. Four-week-old vitamin D-deficient chicks were divided into four groups: Group A served as the normal group and received subcutaneous injections of cholecalciferol; Groups B and C were vitamin D-deficient hearts but perfused differently; Group D received daily subcutaneous injections of 5 micrograms of 1,25(OH)2D3. When the isolated spontaneously beating hearts (modified Langendorff preparation) were perfused with Krebs-Henseleit (KH) solution containing a calcium concentration of 2.5mM, the myocardial contractility of the vitamin D-deficient hearts was significantly increased when compared with group A. After the isolated heart had beaten for one hour, the myocardial contractility in the vitamin D-deficient hearts was found to decline to significantly lower values. Presacrifice administration of 1,25(OH)2D3 improved cardiac performance. Vitamin D deficiency resulted in an enhanced rate of decline of the intracellular high-energy phosphorus compounds. No differences were found in the microscopic study. These observations suggest that vitamin D has a role in cardiac function.