Pathogenesis of fever in delayed hypersensitivity: factors influencing release of pyrogen-inducing lymphokines.

In continuing studies on the pathogenesis of fever in states of delayed hypersensitivity, we have investigated the conditions for the release of an endogenous pyrogen (EP)-inducing lymphokine from draining-lymph-node lymphocytes of rabbits with delayed hypersensitivity to bovine gamma globulin. Using doses of 4 X 10(7) to 5 X 10(7) blood leukocytes (BL) as a source of EP, we found that ratios of about 5:1 of viable lymphocytes to BL were required to stimulate the BL to produce detectable amounts of EP in vitro. Both irradiated lymphocytes (1,700 R) as well as those from steroid-treated donors retained their ability to activate BL when incubated with antigen, properties consistent with activated "T" lymphocytes. In experiments to determine effects of temperature and duration of incubation on lymphokine release, the maximum EP-releasing activity was found to be present in supernatants of sensitized lymphocytes incubated with antigen for 18 h at 37 degrees C. These studies have confirmed that sensitized lymphocytes release a soluble, pyrogen-inducing lymphokine when incubated with antigen and further demonstrate that tissue macrophages (Kupffer cells) as well as BL can be activated to produce EP in vitro by this agent.     

Basic isoferritin and hypercalcaemia in renal cell carcinoma

A 63-year-old man with iron loss anaemia and hypercalcaemia was found to have a renal cell carcinoma. Despite the iron-deficient blood and bone marrow picture, the serum ferritin concentration was markedly raised. This was mainly due to a “basic isoferritin”. The serum parathormone concentration was normal. The serum ferritin and calcium concentrations returned to normal after the tumour was removed. We propose that the renal cell carcinoma cells in this patient secreted the basic isoferritin as well as humoral factor(s) responsible for hypercalcaemia.     

IL-1 up-regulates osteopontin expression in experimental crescentic glomerulonephritis in the rat

Osteopontin (OPN) is a macrophage chemotactic and adhesion molecule that acts to promote macrophage infiltration in rat anti-glomerular basement membrane (GBM) glomerulonephritis. The present study investigated the role of interleukin-1 (IL-1) in the up-regulation of renal OPN expression in this disease model. Accelerated anti-GBM glomerulonephritis was induced in groups of six rats. Animals were treated by a constant infusion of the IL-1 receptor antagonist or saline (control) over days -1 to 14 (induction phase) or days 7 to 21 (established disease). In normal rat kidney, OPN was expressed in a few tubules (<5%) and absent from glomeruli. During the development of rat anti-GBM disease (days 7 to 21), there was substantial up-regulation of OPN mRNA and protein expression in glomeruli (>5 cells per glomerular cross-section) and tubular epithelial cells (50-75% OPN-positive). Up-regulation of OPN expression was associated with macrophage accumulation within the kidney, severe proteinuria, loss of renal function, and severe histological damage including glomerular crescentic formation and tubulointerstitial fibrosis. In contrast, IL-1 receptor antagonist treatment of either the induction phase of disease or established disease significantly reduced OPN mRNA and protein expression in glomeruli (/75-85%, P < 0.001) and tubules (/45-60%, P < 0.001). The reduction in OPN expression was associated with significant inhibition of macrophage accumulation and progressive renal injury. In vitro, the addition of IL-1 to the normal rat tubular epithelial cell line NRK52E up-regulated OPN mRNA and protein levels, an effect that was dose-dependent and inhibited by the addition of IL-1 receptor antagonist, thus demonstrating that IL-1 can act directly to up-regulate renal OPN expression. In conclusion, this study provides in vivo and in vitro evidence that IL-1 up-regulates OPN expression in experimental kidney disease and support for the argument that inhibition of OPN expression is one mechanism by which IL-1 receptor antagonist treatment suppresses macrophage-mediated renal injury.     

Lectin binding to formalin-fixed paraffin sections.

Lectins are potentially useful tools in histopathology for the identification of carbohydrates and distinguishing cells according to their type, differentiation or function. Conjugated to fluorescent or enzyme labels, lectins are simple to use on fresh tissue but fixation and processing sequesters glycoconjugates and dissolves out fat-linked sugars. We describe here the use of labelled antibodies to lectins to localise sites of lectin binding and increase sensitivity, combined with trypsin and neuraminidase to reveal sequestered carbohydrates. Absorbing lectins with appropriate sugars establishes the specificity of binding and allows lectins to be used as sensitive and specific reagents.     

Regulation of the synthesis of Sindbis virus-specified RNA: role of the virion core protein

Cells infected with seven different RNA+ mutants of Sindbis virus were found to accumulate a virus-specified polypeptide of mol. wt. 144000 (p144) during incubation at the non-permissive temperature, while at the same time synthesis of the virus structural proteins was drastically reduced. Mapping of the tryptic peptides of p144 showed that it contained the amino acid sequences of all the virus structural proteins. At the non-permissive temperature cells infected with the same seven mutants (out of 28 examined) also showed increased synthesis of 26S RNA, the mRNA for the virus structural proteins, relative to 42S RNA, and the virus genome, compared with infections by wild-type virus. We propose that both these phenotypic effects are the results of a single mutational step and that the primary defect in the processing of the virus structural protein precursor induces the relatively increased rate of synthesis of structural protein mRNA. Temperature-shift experiments with mutant-infected cells showed that p144 itself is not the agent of this effect. The failure of exposure to zinc ions to alter the RNA ratio in wild-type virus-infected cells suggested that the virus envelope proteins are not involved either, since their synthesis is preferentially inhibited under these circumstances. It is possible that it is the failure to synthesize the proper quantity of core protein in the mutant-infected cells which causes the shift of RNA synthesis in favour of structural protein mRNA.     

Double-blind, placebo-controlled food challenge (DBPCFC) as an office procedure: a manual

There is now enough experience with the use of double-blind, placebo-controlled, food challenge (DBPCFC) to recommend its use as an office procedure for most patients complaining of adverse reactions to foods. This manual discusses the practical methods required for the allergist to undertake DBPCFC in the office. Thorough histories supplemented by food allergen skin testing are used to design a DBPCFC that carefully attempts to reproduce the history of food-induced symptoms described by the patient. Precautions that must be taken are delineated before challenge, as is treatment that may be required if a reaction occurs. For those foods to which challenges are positive, longitudinal evaluation with repeated challenge at appropriate intervals help to determine whether or not the problem will resolve over a period of time.     

Elastin point mutations cause an obstructive vascular disease, supravalvular aortic stenosis

Supravalvular aortic stenosis (SVAS) is an inherited obstructive vascular disease that affects the aorta, carotid, coronary and pulmonary arteries. Previous molecular genetic data have led to the hypothesis that SVAS results from mutations in the elastin gene, ELN. In these studies, the disease phenotype was linked to gross DNA rearrangements (35 and 85 kb deletions and a translocation) in three SVAS families. However, gross rearrangements of ELN have not been identified in most cases of autosomal dominant SVAS. To define the spectrum of ELN mutations responsible for this disorder, we refined the genomic structure of human ELN and used this information in mutational analyses. ELN point mutations co-segregate with the disease in four familial cases and are associated with SVAS in three sporadic cases. Two of the mutations are nonsense, one is a single base pair deletion and four are splice site mutations. In one sporadic case, the mutation arose de novo. These data demonstrate that point mutations of ELN cause autosomal dominant SVAS.      

Morphological analysis of degeneration and regeneration of syncytiotrophoblast in first trimester placental villi during organ culture

We have recently shown using dansyl-L-lysine exclusion studies that the release of human chorionic gonadotrophin (HCG) in conjunction with L- lactate dehydrogenase (LDH) from first trimester villi during organ culture is symptomatic of syncytiotrophoblast degeneration. The purpose of this study was to examine chorionic villi at the ultrastructural level in order to determine events occurring during organ culture. The tissue was sampled after 0, 24, 48 and 120 h in culture and processed for electron microscopy. In addition to confirming the previously recorded syncytial degeneration, the electron micrographs showed clearly the generation of a new syncytiotrophoblast layer. The new layer, derived from differentiating cytotrophoblast cells, was largely formed by 48 h and was maintained for at least 120 h in culture. This study demonstrates a model which provides an opportunity to study the differentiation of cytotrophoblast cells whilst they retain their anatomical relationships within the villous structure.