This month in J Lab Clin Med

The Journal of LABORATORY and CLINICAL MEDICINE Copyright © 1999 by Mosby, Inc. VOLUME 133

APRIL 1999

NUMBER 4

THIS MONTH IN J Lab Clin Med Issue Highlights for April 1999

Iron and infections For over 20 years it has been known that certain infections are more common in a state of iron excess. This was partly because of the ability of iron to act as a growth-promoting agent for certain organisms; in some cases it appeared that chelation therapy might worsen the problem, at least transiently, because iron-laden chelators could act as nutrient siderophores. Since the work of van Asbeck and others in the early 1980s it has been clear that some patients with iron overload also had neutrophil dysfunction--presumably on the basis of iron-catalyzed oxidant injury to phagocytes. Moreover, neutrophils from iron-overloaded patients have been found to contain more iron that do neutrophils from normal subjects. Dr B~ Cantineaux and colleagues from the several University-affiliated hospitals in Brussels studied the ability of plasma from patients with iron overload to produce dysfunction of phagocytes from normal donors. That is, was the dysfunction something that slowly accrued to the cells through continuous oxidant stress over their life spans, or was it something that happened relatively abruptly? As described beginning on page 353, neutrophils from healthy donors were harvested and were incubated with various concentrations of iron citrate or with serum fractions derived from normal subjects or from thalassemic patients with iron excess. The cells were submaximally stimulated with phorbol myristate acetate during the incubation. After the incubation they were tested for their phagocytic capacity with a bacterial target. In this system, incubation with serum from thalassemic patients resulted in a phagocytic defect (when compared with serum-free incubations or incubations with normal serum); this could be prevented by the addition of the iron chelator deferoxamine to the incubation. When serum was fractionated by gel filtration, the fraction containing albumin and transferrin worked no ill effect on neutrophils. In contrast, the fraction containing ferritin produced a phagocytic d e f e c t - - a n d this was more pronounced if the serum had come from an iron-overloaded patient than if it had come from a normal donor. The addition of deferoxamine or catalase (or both) to the incubation lessened the resultant injury. Finally, neutrophils that had been incubated with the ferritin fraction of thalassemic serum were found to contain more L-subunit ferritin than did cells from other incubations. The authors conclude that at least some of the phagocytic defect associated with iron overload is related to exposure to ferritin iron, that it may be seen even after a relatively brief exposure (at least if the cells are actively producing their own oxidants), and that it is associated with ferritin uptake.

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Review: Myeloperoxidase as an important mediator of tissue injury Myeloperoxidase (MPO) deficiency has long been known as a relatively benign neutrophil disorder in which the propensity to infection is limited and often mild enough to be clinically silent. In textbooks, it is often described in terms of what it teaches us in a negative fashion-that the generation of highly reactive oxidants by other systems is biologically more important than is MPO in host defense. That being said, MPO is now known to be involved in the generation of a number of longeracting oxidants (eg, hypohalous acids), which might be important in other contexts, such as post-inflammatory repair or the oxidative modeling of lipids (to good or ill). This month we are pleased to present a review article by Dr Jay Heinecke, of Washington University in St Louis, describing the potential contributions of MPO to inflammatory injury, with a special emphasis on a likely rrle in atherosclerosis. The latter gains credence because substances altered by the action of hypohalous acids and tyrosyl radical can be identified by mass spectroscopy of material in atherotic lesions--a convincing "footprint" of MPO. The author reviews the basic enzymology of MPO, the nature of the "targets" for MPOaugmented oxidation, and the detection of the stable end products in arterial plaque. This review may be found beginning on page 321.

Midkine in the gut Midkine is a heparin-binding growth factor that was first recognized about a decade ago in murine carcinoma cells. Its importance to normal (non-cancerous) homeostasis is still being explored; however, like many of its kin, it shows some ability to promote fibroblast growth and is therefore postulated to have some rrle to play in wound healing. The gene has now been cloned, allowing sensitive examination of midkine expression in various tissues, including under circumstances of wound heating. Dr Toru Maekawa of Kyoto University, with associates from Kobe (Kanebo Memorial Hospital), Osaka (Saiseikai Noe Hospital), and Izumo (Shimane Medical University), studied midkine expression in the rat (see page 349). In the unstressed stomach, they found midkine gene expression in the submucosa and muscularis, areas where fibroblasts are plentiful. They then induced shallow ulcerations of the gastric mucosa by the systemic administration of indomethacin. Typically, these lesions healed fully within a few days, without ever developing a granulation tissue response. These lesions showed no increase in midkine gene or mRNA expression during their evolution or resolution. The investigators then provoked deeper ulcerations, which were slower to heal, by applying acetic acid to the serosal surface of the stomach at laparotomy. The resultant lesions did form granulation tissue en route to healing, with a vigorous fibroblastic response. In contrast to the short-lived nonsteroidal anti-inflammatory drug ulcers, these had prominent midkine gene expression, peaking at about 15 days after the insult. In parallel cell culture studies, midkine was found to be strongly expressed by a (human lung [midline is strongly conserved]) fibroblast cell line, and it was found not to promote the growth of a rattine gastric mucosal cell line. From these studies alone it's hard to be sure whether midkine is the cart or the horse: Is it there because fibroblasts make it, or is it there because it is stimulating fibroblastic response? Knowing from other studies that midkine promotes fibroblast growth, one might very well answer, "Both." That is, it may be part of an autocrine/paracrine stimulus/response network that promotes vigorous fibroblast growth in response to an injury that requires a repair that quickly develops substantial mechanical strength. If that's the case, another question quickly comes to mind: What turns it off?

J Lab Clin M e d Volume 133, N u m b e r 4

In this issue

Soybeans and the dying liver The intake of large amounts of ethanol has long been known to be a major contributor to liver disease, especially cirrhosis. Some of this damage is probably attributable to the generation of oxidants as by-products as alcohol is metabolized. There is great individual variation in susceptibility to injury by alcohol; among the known additive risk factors are chronic viral hepatitis and iron overload--factors that would be expected to increase the net oxidant burden. It is therefore now of interest to ask whether some of the risk-decreasing factors might be things that would decrease oxidant burden, protect against oxidant injury, or promote the repair of oxidant damage. In 1994 it was reported that a soybean extract rich in phosphatidylcholine protected against hepatic fibrosis in baboons chronically fed ethanol. That observation has now been taken to a cell culture system by Dr Jaime Poniachik et amici from the Bronx VA Medical center and the Mount Sinai School of Medicine (New York), and their findings are presented this month beginning on page 342. Part of the process of cirrhosis is the conversion of hepatic stellate cells to myofibroblastlike cells. This specific change has been found to be inhibited in baboons fed soybean extract, but it was unclear whether the effect was a direct one on these cells. To answer that question, the authors cultured stellate cells under circumstances that promoted their transformation, in the presence and absence of soybean extracts. Conveniently, the transformation is associated with cell proliferation and the production of type I collagen and smooth-muscle actin, providing robust end points to measure (rather than just morphologic change). The original extract used in the baboon studies contained large amounts of dilinolineoylphosphatidylcholine and of palmitoyl-linoleoyl-phosphatidylcholine;it was also unclear from those early studies whether these two lipids both contributed to the protective effect or if the "bang" was attributable to one of them specifically (or indeed to another, less-plentiful component). When low concentrations of dilinolineoyl-phosphatidylcholine were included in stellate cell cultures, thymidine incorporation and the production of procollagen and actin were lower than those in comparison cultures without addition or with only a solvent blank. In contrast, the addition of 1,palmitoyl 2,1inoleoyl phosphatidylcholine at similar concentrations allowed the transformation to proceed unimpeded. Interestingly, the amount of mRNA for type I procollagen was comparable in all incubations, suggesting that the effect of the phospholipid was post-translational. Thus it appears that at least some of the liver-protective effect of soybean phospholipid extract may be due to a direct effect on the tendency of stellate cells to transform under stress to myofibroblast-like (collagen-producing) cells. This effect resides in dilinolineoyl-phosphatidylcholine. Although these studies are still a bit removed from clinical cirrhosis, it is exciting to speculate that the manipulation of dietary lipids and antioxidant status might ere long provide tools to prevent or delay cirrhosis in patients at high risk.

Why do Hell's Angels have long thrombin times? (And why are their protimes sometimes long, too?) When I was a trainee, a member of a motorcycle gang was presented to a hematology conference because of the curious finding of an unclottable thrombin time (TT) with normal prothrombin time (PT) and partial thromboplastin time (PTT). Normally, thrombin inhibition (or fibrinogen lack) sufficient to render the thrombin time unclottable would be expected to prolong the other clotting times substantially as well. We postulated that an alloantibody to the reagent (bovine) thrombin in the TT might be specific enough for the PT and PTT to be unaffected (the thrombin in those assays is gen-

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erated from the patient's plasma and is thus of human origin). We further guessed that such an antibody might have resulted from the use of topical thrombin as a hemostatic agent in (known) prior trauma surgery. Unfortunately, the patient had died by the time of the conference, and we were never able to test our hypothesis in him; however, the occurrence of such alloantibodies has been well documented in many subsequent patients. More recently it has been noted that alloantibodies may occur in response to other proteins that are minor components of the bovine thrombin preparations used as hemostatic agents in reconstructive surgery and trauma surgery. These may include antibodies to other coagulation factors, such as factor IX and factor V. Unfortunately, these antibodies sometimes cross-react with the human analogs well enough to influence coagulation tests and even to cause clinical bleeding. Dr Thomas Ortel of Duke University reviews for us this month the topic of antibodies to factor V, including those arising as cross-reacting antibodies to the bovine factor and autoanfibodies arising spontaneously or in association with drug use. His review may be found beginning on page 326. For the editors Dale E. Hammerschmidt, MD Editor- in- Chief