Hepatocytes in collagen sandwich: evidence for transcriptional and translational regulation.

The influence of extracellular matrix configuration on the tissue-specific function of cultured hepatocytes was investigated. Adult rat hepatocytes sandwiched between two layers of collagen gel were compared to cells cultured on a single layer of collagen gel for differences in the total RNA content, the level of albumin-specific mRNA, the rate of albumin gene transcription, and the rate of albumin mRNA translation. Adult hepatocytes in the sandwich system maintained the level of albumin mRNA similar to that found in the normal liver for at least six weeks, whereas the level of albumin mRNA declined rapidly in the single gel system. After one week of culture, hepatocytes in the single gel system could be induced to recover the high level of albumin mRNA and albumin production when a second layer of collagen gel was overlaid at that time. Furthermore, sandwiched hepatocytes maintained significantly higher transcriptional activity compared to cells in the single gel system. In addition to transcriptional control, the ultimate rate of albumin production was shown to depend on the rate of translation, which increased with culture time and reached a plateau in one to two weeks. This increase in translational activity over time in culture was observed in both the sandwich and the single gel systems and, thus, appeared to be independent of the configuration of extracellular matrix.

Sequential translation of trinucleotide codons for the initiation and termination of protein synthesis.

Terminal events in protein synthesis were studied with trinucleotide codons. Initiator and terminator trinucleotides sequentially stimulate N-formyl-methionyl-tRNA binding to ribosomes and the release of free N-formyl-methionine from the ribosomal intermediate. The release factor discovered by Capecchi is also required. The trinucleotides UGA, UAA, and UAG were found to be terminator codons. This pattern of codon degeneracy has not been observed with other trinucleotides and transfer RNA.

Common virulence factors for bacterial pathogenicity in plants and animals.

A Pseudomonas aeruginosa strain (UCBPP-PA14) is infectious both in an Arabidopsis thaliana leaf infiltration model and in a mouse full-thickness skin burn model. UCBPP-PA14 exhibits ecotype specificity for Arabidopsis, causing a range of symptoms from none to severe in four different ecotypes. In the mouse model, UCBPP-PA14 is as lethal as other well-studied P. aeruginosa strains. Mutations in the UCBPP-PA14 toxA, plcS, and gacA genes resulted in a significant reduction in pathogenicity in both hosts, indicating that these genes encode virulence factors required for the full expression of pathogenicity in both plants and animals.

Relationship of age to incidence of breast cancer in young women.

The rate of increase with age in the incidence of breast cancer in women was at a maximum at the lowest age that it could reasonably be estimated (in these data, 25 yr). It then declined linearly with age to about 50 years. The rate of increase with age, and its changes with age, were similar in many Western populations and in Japan. The decline with age in the rate of increase in the incidence of breast cancer was arrested at the age of 50 and replaced by rates of change that altered little with age. The premenopausal changes could be reproduced by a breast cancer precursor model with exponential decay of precursor prevalence with time. There was evidence for the existence of such a precursor.

Spatial distribution of tumor-specific monoclonal antibodies in human melanoma xenografts.

The time-dependent (1-72-h) spatial distribution of three biotinylated anti-melanoma monoclonal antibodies (MAbs), a control MAb, and several macromolecular tracers was studied in two small (4-12-mg), well-characterized human melanoma xenografts (SK-MEL-2, M21) growing in the s.c. space of athymic nude mice. The specific MAbs (436, IND1, and 9.2.27) recognize two different melanoma cell surface antigens (Mr 125,000 glycoprotein melanoma-associated antigen and high molecular weight melanoma-associated antigen) and have equilibrium association constants differing by two orders of magnitude (10(8)-10(10) M-1). SK-MEL-2 tumors were poorly vascularized and were composed of one or several collections of tumor cells with few intratumor blood vessels. In contrast, M21 tumors induced a strong angiogenic response and were organized into multiple small tumor cell nests separated from each other by fine blood vessels. Neither tumor developed extensive connective tissue stroma. In both tumors, hyperpermeable blood vessels were concentrated at the tumor-host interface but some intratumor vessels in M21 tumors were also leaky. Macromolecular tracers extravasated extensively from leaky vessels into tumor stroma but penetrated poorly into tumor parenchyma. All three tumor-specific MAbs stained tumor cell surfaces in a time-dependent fashion such that one-half or more of all tumor cells were stained by 24-48 h. Tumor cell staining was favored by increased density of tumor cell antigens but, at the doses studied, was little affected by differences in affinity among tumor-specific antibodies. The distribution of MAb staining was nonuniform in two respects: (a) peripherally situated tumor cells were more likely to be stained than centrally placed cells, and only in the smallest tumors did MAb reach centrally placed tumor cells; and (b) staining was nonuniform in different parts of the same tumor. The inhomogeneity of tumor cell staining by tumor-specific MAb was attributable to several factors, including: tumor blood vessel number, distribution, perfusion and permeability; distribution of tumor connective tissue stroma; small volume of the parenchymal interstitial space and relatively impaired diffusion of macromolecules in that space (low effective diffusivity of MAb); and interactions between specific MAbs and tumor cells. Of these factors, those associated with the parenchymal compartment apparently were rate limiting, and strategies that enhance parenchymal penetration are likely to improve solid tumor therapy with MAbs.

A quantitative analysis of tumor specific monoclonal antibody uptake by human melanoma xenografts: effects of antibody immunological properties and tumor antigen expression levels.

The time-dependent (5 min-72 h) localization of 3 radiolabeled anti-melanoma monoclonal antibodies (MAbs 436, IND1, and 9.2.27) was studied in paired label experiments in small (4-12 mg) s.c. human melanoma xenografts (SK-MEL-2 and M21) in athymic nude mice. MAb 436 recognizes a Mr 125,000 cell surface melanoma-associated glycoprotein antigen (125 kDa-MAA); MAbs IND1 and 9.2.27 recognize a high molecular weight melanoma-associated antigen, but with equilibrium association constants differing by 2 orders of magnitude (10(8)-10(10) M-1). The two tumors were found to differ in their antigen expression levels and in both interstitial and vascular volumes. Accumulation of MAbs in both tumors was determined primarily by antigen expression levels and also by physiological factors such as vascular permeability and vascular volume; at the dose administered (20 micrograms/mouse), differences in MAb affinity among specific MAbs had minimal effect on accumulation. Quantitative flow cytometry measurements showed that antigen expression in vivo differed from that of cultured tumor cells. In vivo, expression of the Mr 125,000 MAA decreased by a factor of about 2.5 in both tumors. In contrast, the in vivo expression of the high molecular weight MAA decreased in M21 tumors but increased by 2.0-3.5-fold in SK-MEL-2 tumors. Data were analyzed using a three-compartment pharmacokinetic model (C. Sung et al., Cancer Res., 52:377-384, 1992) to provide plasma-to-tissue transport constants (k), the interstitial fluid flow rate (L), and estimates of the in vivo interstitial MAb binding site concentration (B0). For all MAbs, the plasma-to-tissue transport constants were consistently greater for M21 tumors (0.44-0.85 microliter/min/g) than for SK-MEL-2 tumors (0.28-0.66 microliter/min/g), and values of k for both tumors were approximately 1 order of magnitude greater than those for skeletal muscle (0.06-0.08 microliter/min/g). The model-estimated binding site concentration of melanoma-specific antibodies was 15-70 times lower than that predicted by experimental measurements of tumor antigen concentrations. Factors that may contribute to this discrepancy include inaccessibility of tumor cell binding sites to MAb and MAb catabolism. In summary, these results indicate that, for the MAb dose used in this study, variables pertaining to the tumor target (i.e., antigen expression levels, vascular volume, and vascular permeability) are the most important for determining MAb accumulation in tumors.

Recognition of streptococcal pharyngitis in adults.

In order to devise a strategy for the management of acute pharyngitis, the clinical features of 418 adults with sore throat were noted and throat cultures were obtained. Patients with cultures positive for group A beta-hemolytic streptococci had a significantly higher (P less than or equal to .01) frequency of recent exposure to streptococcal infection, pharyngeal exudate, enlarged or tender cervical nodes, and high fever (greater than or equal to 38.3 C [101 F]. Patients with negative cultures complained more frequently of cough. On the basis of these symptoms and signs, a clinical algorithm was developed and discriminant function scores were computed that identify patient populations with different probabilities of having streptococcal pharyngitis. The patients with moderate and high probabilities included 91% of patients with positive cultures but only 67% of the total patient population. These methods could be the basis for more efficient evaluation of adults with sore throat.

Immobilized IL-2 preserves the viability of an IL-2 dependent cell line.

The mouse T-cell line CTLL-2 is known to be dependent on interleukin-2 (IL-2) for both growth and viability. These cells possess high affinity IL-2 receptors and have been shown to internalize IL-2 after binding. To determine if internalization of IL-2 is required for the mediation of its signal, IL-2 was covalently coupled to an insoluble matrix via glutaraldehyde cross-linking and CTLL-2 cells were incubated with the immobilized lymphokine matrix. This covalent cross-linking prevents the free lateral diffusion and internalization of the bound IL-2 receptors (IL-2R) while still permitting specific binding between the cells and the immobilized ligands. Although only very limited proliferation was observed during the incubation as assessed by 3H-thymidine incorporation, the viability of the CTLL-2 cells on the immobilized IL-2 matrix was preserved. Cells incubated on the immobilized IL-2 surface could proliferate in response to exogenous soluble IL-2 that was added to the cultures after 36 hours whereas control cultures incubated with an immobilized BSA matrix had died. This indicates that immobilized IL-2 can mediate some of the activity of soluble IL-2 and that internalization of the IL-2 receptor may not be required for at least part of the IL-2 mediated effect.

Genetically modified human keratinocytes overexpressing PDGF-A enhance the performance of a composite skin graft.

Skin loss due to burns and ulcers is a major medical problem. Bioengineered skin substitutes that use cultured keratinocytes as an epidermal layer with or without analogues of the dermis are one strategy for skin repair. However, none can achieve definitive wound closure, function, or cosmesis comparable to split-thickness autografts. Moreover, autograft donor sites, which require time to heal, may be limited or have attendant problems such as infection or functional/cosmetic deficiencies. To determine if the performance of composite skin grafts of keratinocytes on a dermal analogue could be enhanced, human keratinocytes were genetically modified to overexpress platelet-derived growth factor A chain (PDGF-A). Composite grafts of modified keratinocytes seeded onto acellular dermis, prepared from cryopreserved cadaver skin, secreted PDGF-AA protein in vitro [90 ng/graft (1.5 x 1.5 cm)/24 hr]. To test their performance in a wound healing model, composite grafts were transplanted to full-thickness excisional wounds on the back of athymic mice. PDGF-A grafts formed a stratified differentiated epidermis similar to control grafts. The acellular dermis was repopulated with host fibrovascular cells and by day 7, the PDGF-A grafts had significantly more cells in the dermis and increased staining for murine collagen types I and IV. At this early time point, wound contraction was also significantly inhibited in PDGF-A grafts versus control grafts. Thus, PDGF-A overexpression improves graft performance during the first critical week after transplantation.

Characterization of a composite tissue model that supports clonal growth of human melanocytes in vitro and in vivo.

To aid in the investigation of factors that control the proliferation and function of melanocytes, we have characterized a skin equivalent model that supports melanocyte growth and function in vitro and in vivo. Passenger melanocytes survive and proliferate at low numbers when keratinocytes of the epidermis are cultured in serum-containing medium using a fibroblast feeder layer. When the surface of de-epidermalized acellular dermis was seeded with these cultured cells, the keratinocytes formed a stratified epithelium in vitro containing rete ridges, and the melanocytes were preferentially located in the bottom of these rete ridges. Melanocyte cell number was much less than in normal skin, but in some areas the melanocytes were in clusters, consistent with clonal growth of the cells. When transplanted to athymic mice, the grafts formed foci of pigmentation at 3 wk that expanded and repigmented the entire graft by 8 wk. Histologic examination of these foci revealed that they corresponded to clusters of melanocytes that proliferated and migrated to eventually repopulate the entire graft. In grafts of mixed cells from light and dark skin donors, distinct foci of pigmentation were obvious at 3 wk and, instead of progressing to complete repigmentation, these foci remained stable for over 6 wk. Histologic examination confirmed that these grafts of mixed cells were entirely repopulated with melanocytes and that the grafts contained distinct zones of melanocytes that were of exclusively dark or light skin origin. This model should be valuable for studying the clonal growth of melanocytes in the context of the epidermis.

Evaluation of human skin reconstituted from composite grafts of cultured keratinocytes and human acellular dermis transplanted to athymic mice.

This study evaluates the use of composite grafts of cultured human keratinocytes and de-epidermalized, acellular human dermis to close full-thickness wounds in athymic mice. Grafts were transplanted onto athymic mice and studied up to 8 wk. Graft take was excellent, with no instances of infection or graft loss. By 1 wk, the human keratinocytes had formed a stratified epidermis that was fused with mouse epithelium, and by 8 wk the grafts resembled human skin and could be freely moved over the mouse dorsum. Immunostaining for keratins 10 and 16 and for involucrin revealed an initial pattern of epithelial immaturity, which by 8 wk had normalized to that of mature unwounded epithelium. Mouse fibroblasts began to infiltrate the acellular dermis as early as 1 wk. By 8 wk fibroblasts had completely repopulated the dermis, and blood vessels were evident in the most superficial papillary projections. Dermal elements, such as rete ridges and elastin fibers, which were present in the starting dermis, persisted for the duration of the experiment. Grafts using keratinocytes from dark-skinned donors as opposed to light-skin donors had foci of pigmentation as early as 1 wk that progressed to homogenous pigmentation of the graft by 6 wk. These results indicate that melanocytes that persist in vitro are able to resume normal function in vivo. Our study demonstrates that composite grafts of cultured keratinocytes combined with acellular dermis are a useful approach for the closure of full-thickness wounds.

Genetically modified human epidermis overexpressing PDGF-A directs the development of a cellular and vascular connective tissue stroma when transplanted to athymic mice--implications for the use of genetically modified keratinocytes to modulate dermal regeneration.

We investigated the hypothesis that keratinocyte-produced platelet-derived growth factor-AA (PDGF-AA) is involved in epidermal-dermal interactions and that PDGF-AA is an important mediator of the temporal and spatial events of tissue repair. Retroviral-mediated gene transfer was used to introduce the gene encoding human PDGF-A into cultures of human diploid keratinocytes. Genetic modification boosted the endogenous in vitro level of PDGF-AA secretion by over 300 fold. When PDGF-secreting cells were transplanted as epithelial sheets to athymic mice, modified keratinocytes underwent terminal differentiation and generated a stratified epithelium comparable to unmodified cells. Seven days after grafting the newly synthesized connective tissue layer subjacent to the PDGF-A-modified grafts was significantly thicker, was rich in mononuclear cells and fibroblasts, and had increased numbers of blood vessels when compared to control grafts of unmodified cells. These results suggest that PDGF-AA secreted by the epidermis is an important mediator of epithelial-mesenchymal interactions and helps to promote growth and vascularization of the underlying dermal tissue. Further, these data demonstrate the feasibility of using genetically modified cells to modulate tissue regeneration.

Electroconvulsive therapy and memory dysfunction: is there evidence for prolonged defects?

The authors reviewed 39 papers which concern the long-term effects of electroconvulsive therapy (ECT) on human memory. Although the authors caution that methodological considerations preclude a decisive assessment, the majority of the studies suggest that ECT does not normally produce prolonged memory defects. Some recent studies do document subtle but persistent defects several months after ECT, especially in personal autobiographical material. These defects appear to be more annoying than seriously incapacitating. Variables considered important in an ideal design of studies on ECT and memory are discussed.

Clinical correlates of lateral ventricular enlargement in bipolar affective disorder.

The presence of lateral ventricular enlargement in some manic-depressive subjects, as assessed by ventricular-brain ratios (VBRs), has been reported. A study of 27 bipolar patients and 27 individually matched normal controls confirmed that finding. Bipolar patients had significantly larger VBRs than did controls. Clinical measures associated with the presence of ventricular enlargement in the bipolar patients included more frequent hospitalizations and histories of persistent unemployment. Other measures of illness severity or social deterioration were not significantly associated with large VBR.

Effect of tetraploidy on dendritic branching in neurons and glial cells of the frog, Xenopus laevis.

Morphological aspects of four different groups of Golgi impregnated brain cells from a tetraploid strain of Xenopus laevis frogs were compared to analogous cells in comparably sized diploid frogs. The cells examined included neurons from the telencephalon, caudal hypothalamus, and optic tectum, and radial glial cells from the optic tectum. The brains of tetraploid frogs appeared grossly normal and were the same size and contained similar cell types as diploid brains. As observed in previous studies on polyploid amphibia, somal diameters increased significantly in tetraploid cells for each of the four groups of cells examined. Also, the total length of the dendritic arbors in tetraploid brain cells increased significantly by factors ranging from 1.4 to 2.4 times the total length of the analogous processes in diploid cells. Tetraploid neurons in the telencephalon and hypothalamus increased their arbor lengths predominantly by increasing the number of dendritic branches, while maintaining the average distance between branch points in the dendritic segments. In contrast, the tetraploid large pear-shaped neurons in the optic tectum had significantly longer terminal dendritic segments than the analogous diploid neurons, although these tetraploid neurons maintained their average number of dendritic segments per cell. Tetraploid tectal radial glial cells appeared to increase both their number of branches and the lengths of their terminal segments. Thus, the mode by which tetraploid brain cells achieved longer dendritic arbors varied from cell type to cell type. These results suggest a hypothetical basis for possible effects of genomic size on vertebrate brain structure and evolution at the cellular level.

Neuroanatomy of Spastic, a behavior mutant of the mexican axoloti: Purkinje cell distribution in the adult cerebellum.

The spastic mutant, found in the Mexican axolotl, shows swimming coordination and equilibrium deficiencies. Histological analyses of wild-type and spastic mutant cerebella previously characterized in physiological studies revealed changes in Purkinje cell location in the mutant auricle or vestibulo-cerebellum. Purkinje cells are "translocated" ventrally correlated with a similar translocation of vestibular single units described previuosly (Ide, '77). Where wild-type Purkinje cells are distributed from the surface to a depth of 250 micrometers, mutant Purkinje cells are "crowded" between 250 and 350 micrometers. Although mutant granule cells are present, boundaries between granule cell and Purkinje cell zones are less precise in mutants. Cerebellar nucleus cells are translocated medially, failing to organize into the discrete cell group appearing in wild-type. Cerebellar white matter tracts and fibers show changes, both in orientation with respect to the underlying tegmentum, and in fascicular organization. Obvious changes in the gross anatomy of the cerebellum are confirmed in reconstructions which define cell and fiber translocation. Thus, the spastic gene is compatible with differentiation of all cerebellar elements, but appears to alter interactions between cells, or between cells and the external milieu. Although all cell types are present in the mutant cerebellum, they fail to attain their proper positions along all three body axes.

The development of retinal ganglion cells in a tetraploid strain of Xenopus laevis: a morphological study utilizing intracellular dye injection.

The morphological development of retinal ganglion cells was examined in a tetraploid strain of Xenopus frogs. The enlarged cells of the tetraploid strain facilitate the application of intracellular techniques. Using an in vitro retinal preparation and Nomarski optics, intracellular recording and dye injection were carried out under visual control on ganglion cells in central retina from 2 days of development (stage 24) to metamorphosis (stage 64). We identified three phases in the morphological differentiation of ganglion cells. During the first phase (stages 24-30), all cells were neuroepitheliallike in form and possessed robust resting potentials in the range of -35 to -60 mV, and dye-coupling was occasionally observed between neighboring cells. During the second phase of ganglion cell development (stages 31-45) the neurons had begun to elaborate axons and dendrites. These cells possessing neurites had resting potentials between -15 and -30 mV, and no dye-coupling was observed between neighbors. During the third and final phase of maturation, from stage 46 onward, three distinct morphological types of ganglion cells could be identified. Type I cells had the smallest somata and the smallest-diameter dendritic arborizations. The profusely branched dendrites of these cells ramify extensively throughout the inner plexiform layer. Type II cells had large somata, intermediate-diameter dendritic fields, and a highly elaborate dendritic branching pattern. These cells were seen to arborize within two sublamina in the inner plexiform layer. Type III cells had large somata, the largest-diameter dendritic fields, and a dendritic arbor with long primary branches but little higher-order branching. These large dendritic fields were confined to a single sublamina of the inner plexiform layer, abutting the inner nuclear layer. While most phase 3 cells showed radial axon trajectories from the soma to the optic disc, a minority of cells (1-5%) with erratic and nonradial axon trajectories were also observed. Our data provide a morphological description of ganglion cell maturation in the central retina of Xenopus. We show that very early in development (as early as stage 46) three distinct morphological types of retinal ganglion cells are present, which correspond to the three classes of ganglion cells previously described in adult Xenopus (Chung et al., '75).

Relations among arginine, citrulline, ornithine, and leucine kinetics in adult burn patients.

Plasma fluxes of arginine, citrulline, and leucine, and the rate of conversion of labeled citrulline to arginine (Qcit-->arg) were determined in nine severely burned patients (mean: 56% body surface burn area, mean 10 d postinjury) while they received total parenteral nutrition (TPN) including an L-amino acid mixture that supplied a generous amount of nitrogen (mean: 0.39 +/- 0.02 g.kg-1.d-1). Plasma fluxes were also studied in these patients during a basal state (low-dose intravenous glucose) by using a primed, 4-h constant intravenous tracer-infusion protocol. Stable-nuclide labeled tracers were L-[15N-15N-guanidino,5,5,2H2]arginine; L-[13C-ureido]citrulline; L-[1-13C]leucine; and NaH13CO3 (prime only), with blood and expired air samples drawn at intervals to determine isotopic abundance of arginine, citrulline, ornithine, and alpha-ketoisocaproate (KIC; for leucine) in plasma and 13CO2 in breath. Leucine kinetics (flux and disappearance into protein synthesis) confirmed the anticipated higher protein turnover in these burn patients compared with healthy control subjects. The plasma arginine fluxes were correspondingly higher in burn patients than in healthy control subjects. However, the citrulline flux and rate of conversion of citrulline to arginine were not higher than values obtained in our laboratories in healthy adult subjects. We hypothesize that the higher rates of arginine loss from the body after burn injury would need to be balanced by an appropriate exogenous intake of preformed arginine to maintain protein homeostasis and promote recovery from this catabolic condition.

Kinetics of plasma arginine and leucine in pediatric burn patients.

The dynamic status of whole-body arginine and leucine was investigated in eight severely burned (mean 55% of body surface area) pediatric patients (mean age 5.3 y) at a mean of 16 d after their initial injury. Plasma amino acid kinetics were estimated by using primed constant intravenous infusions of L-[13C-guanidino]arginine and L-[I-13C]leucine given for 4 h. Each patient was studied twice within 2 d. The patients were studied either in a "basal" state, which involved removal of amino acids from the total parenteral nutrition (TPN) solution for 8 h before the tracer study, or while receiving complete TPN. Nitrogen intake was 0.58 +/- 0.08 g.kg-1.d-1 with nonprotein energy intake equivalent to 197 +/- 29 kJ.kg-1.d-1. Plasma leucine and arginine fluxes (mumol.kg-1.h-1) were 208 +/- 35 and 108 +/- 18 for basal and 290 +/- 38 and 195 +/- 22 for TPN periods, respectively. Leucine oxidation was 42 +/- 7 and 59 +/- 9 mumol.kg-1.h-1 for basal and TPN periods, respectively, indicating a higher rate of leucine loss in the absence of a leucine intake than that expected for healthy individuals. The arginine kinetic data implied little net de novo arginine synthesis and further suggested increased rates of arginine degradation from burn injury. The expected rate of urea excretion, based on the basal rate of leucine oxidation, agreed closely with the measured output of urinary urea. These findings suggest that arginine is a conditionally indispensable amino acid for maintaining body protein homeostasis and nutrition in severely burned pediatric patients. The metabolic response of these children appears to be quantitatively similar to that for severely burned adult patients.

Imaging infections with antibodies. A quantitative autoradiographic analysis.

Radiolabeled IgG has recently been demonstrated to effectively image infections. A potential but unproven mechanism for this localization is the specific binding of IgG to Fc receptors on the surface of inflammatory cells in infections. In an animal model of soft tissue infection, quantitative autoradiography was used to measure 125I-labeled IgG and albumin in tissues with a spatial resolution sufficient to associate these proteins with cellular morphology. Gamma camera images at 24 h localized the infection with target-to-background ratios of 2.2 +/- 0.5 for IgG and 2.3 +/- 1.0 for albumin (mean +/- SD). Using quantitative autoradiography at 1 h post-injection, significantly higher concentrations were found in infected thighs of 2-4% of initial plasma concentrations (CPo) as compared to 0.2-0.3% of CPo in noninfected thighs (P less than 0.05); at 24 h post-injection, higher concentrations (7-8% of CPo) were found in infected thighs. Radiolabeled proteins were not inflammatory cell associated and were localized primarily within the edematous interstitial spaces of the infection.