Enhanced de novo alloantibody and antibody-mediated injury in rhesus macaques.

Chronic allograft rejection is a major impediment to long-term transplant success. Humoral immune responses to alloantigens are a growing clinical problem in transplantation, with mounting evidence associating alloantibodies with the development of chronic rejection. Nearly a third of transplant recipients develop de novo antibodies, for which no established therapies are effective at preventing or eliminating, highlighting the need for a nonhuman primate model of antibody-mediated rejection. In this report, we demonstrate that depletion using anti-CD3 immunotoxin (IT) combined with maintenance immunosuppression that included tacrolimus with or without alefacept reliably prolonged renal allograft survival in rhesus monkeys. In these animals, a preferential skewing toward CD4 repopulation and proliferation was observed, particularly with the addition of alefacept. Furthermore, alefacept-treated animals demonstrated increased alloantibody production (100%) and morphologic features of antibody-mediated injury. In vitro, alefacept was found to enhance CD4 effector memory T cell proliferation. In conclusion, alefacept administration after depletion and with tacrolimus promotes a CD4+memory T cell and alloantibody response, with morphologic changes reflecting antibody-mediated allograft injury. Early and consistent de novo alloantibody production with associated histological changes makes this nonhuman primate model an attractive candidate for evaluating targeted therapeutics.

Clinical presentation, natural history, and treatment of crescentic proliferative IgA nephropathy.

IgA nephropathy is one of the most common causes of glomerulonephritis in the world and is characterized histologically by the deposition of polymeric forms of IgA within the mesangium and in some cases along the glomerular capillary wall.(1) Proliferative and crescenteric forms of IgA are associated with nephrotic range proteinuria, accelerated hypertension, and a more rapid decline toward end-stage renal disease. Previous attempts to categorize the incidence and clinical significance of proliferative IgA nephropathy have given conflicting results. This is in part the result of the lack of a uniform nomenclature and the failure of clinical therapies to prolong renal survival in specific subgroups. In the present study, we performed a prospective open-label trial of pulse solumedrol and intravenous cyclophosphamide in 20 patients with IgA nephropathy and at least 10% cellular crescents or endocapillary proliferation on renal biopsy. Seventeen patients underwent repeat kidney biopsies after 6 months of therapy, and the morphologic response to treatment was assessed using a modified systemic lupus erythematosis (SLE) histologic activity and chronicity index score. To determine the long-term efficacy of intravenous cyclophosphamide on renal survival, the results of the treated patients were compared with 12 untreated historical controls. Pulse solumedrol and intravenous cyclophosphamide effectively reduced peak serum creatinine, degree of proteinuria, the rate of decline in renal function, and the incidence of end-stage renal disease at 36 months.

Clinicopathologic features of nephrogenic rests and nephroblastomatosis.

Nephrogenic rests are the consequence of residual metanephric tissue in a fully developed kidney. They usually occur along the perimeter of a mature renal lobe (i.e., perilobar), within the lobe itself (i.e., intralobar), or both (i.e., combined). Nephrogenic rests can be grossly obvious or microscopically discrete. Nephroblastomatosis designates nephrogenic rests that are multifocal or diffuse, and implies more extensive disease. Universal (panlobar) nephroblastomatosis denotes complete replacement of the renal lobe by nephrogenic tissue. The fate of nephrogenic rests and nephroblastomatosis varies and includes obsolescence, sclerosis, dormancy, hyperplasia, or neoplasia. Evidence strongly suggests that neoplastic transformation of nephrogenic rests results in Wilms' tumor (nephroblastoma). Nephrogenic rests almost always occur in the setting of Wilms' tumor; perilobar rests show a strong association with synchronous bilateral Wilms' tumors, whereas intralobar rests are more strongly associated with metachronous tumors. Genetic studies have shown that nephrogenic rests often share many of the same chromosomal defects as Wilms' tumor, which provides further evidence that they are precursors to nephroblastoma. Thus, nephrogenic rests are recognized as clinically significant entities requiring adequate detection and close surveillance. Heightened awareness regarding the clinical relevance of nephrogenic rests and nephroblastomatosis (1) has led to improved detection of these precancerous lesions, (2) fostered more intensive investigation into their biologic behavior, and (3) initiated in-depth discussions about potentially new treatment regimens. The pathologists' ability to identify and detect nephrogenic rests has benefited from the more efficient Beckwith classification. Radiologists have deployed high-resolution radiologic/imaging modalities to improve detection of nephrogenic rests in situ. Clinicians and surgeons are more aware of the impact that nephrogenic rests have upon patient management. Despite this progress, more data is needed to further define these lesions.

Characterization of fatty acid synthase in cell lines derived from experimental mammary tumors.

The lipogenic enzyme fatty acid synthase (FAS) is elevated in various human primary cancers and certain human cancer cell lines. FAS overexpression in human neoplasia has clinical relevance because of its association with tumor aggression and potential chemotherapeutic intervention. Here, we surveyed FAS in cell lines established from normal murine mammary epithelium (NMuMG) and from mammary tumors induced by either rodent polyoma (Py) virus or murine mammary tumor virus (MMTV). Western blotting revealed greater content of FAS in Py-transformed A1-1 and T1 than NMuMG or MMTV-transformed Mm5MT, RIIIMT and MMT060562. These data suggest that signaling events mediated by Py transformation may increase cellular amounts of FAS. Although FAS content was elevated to similar levels in A1-1 and T1, specific activities were significantly different as enzyme activity in T1 was 3-fold higher than A1-1. Likewise, FAS activity in NMuMG was about 0.5-fold higher than the MMTV-transformed lines, even though enzyme content was similar. Immunoprecipitation studies employing anti-phosphoamino acid antibodies followed by immunoblot analysis with anti-FAS antisera (and vice versa) were used to characterize the constitutive phosphorylation state of the enzyme. Phosphoserine and phosphothreonine residues were detected in the more active FAS from T1 and NMuMG, but not in the less active FAS from Mm5MT or A1-1. Discovery of phosphorylated FAS suggests that the enzyme may have more immediate control over lipogenesis than previously thought. High-dose (10-4 M) dexamethasone induced FAS content and activity in NMuMG and MMTV-transformed lines but not Py-transformed cells. Lower concentrations (10-8, 10-6 M) of dexamethasone also activated FAS but without concomitant elevation of its protein content, which was consistent with a phosphorylated form of FAS. Finally, cell lines were treated with the FAS inhibitor cerulenin: almost all breast cancer lines were growth inhibited at significantly lower amounts of drug than normal cell lineages, suggesting that FAS plays a greater role in viability of tumor cells than normal cells. Pretreatment with palmitate (a primary end-product of FAS) prior to cerulenin rescued A1-1 cells only slightly from growth inhibition, whereas pretreatment with oleate (a monounsaturated fatty acid synthesized from palmitate) synergized cerulenin's cytotoxic effects.

Increased expression of fatty acid synthase (OA-519) in ovarian neoplasms predicts shorter survival.

Certain cancers exhibit derangement of de novo fatty acid biosynthesis, manifested as overexpression and hyperactivity of the lipogenic enzyme fatty acid synthase (FAS). Correlation of elevated FAS with high tumor grade and advanced stage in primary breast, prostate, and colorectal cancers has drawn attention to the enzyme as a possible marker of poor prognosis. To find a similar utility of FAS in ovarian neoplasms, we compared FAS expression in 68 ovarian tumors with their histological features and clinical outcome. Immunohistochemical localization of FAS was observed in 48 (71%) cases in which staining was either focal (defined as positive staining in 1% to 20% of cells) or multifocal/diffuse (positive staining in >20% of cells). Most (83%) of the 48 cases were represented by endometrioid, serous, or mucinous carcinomas and malignant mixed mullerian tumors (MMMTs). In contrast, ovarian adenomas and tumors of low malignant potential (LMPs) contained little or no FAS. Association between FAS expression and histological diagnosis was statistically significant. The extent of FAS immunostaining was also predictive of prognosis. Among all patients with ovarian malignancies (including LMPs), median survival was 64.8 months, when their tumors exhibited no or focal immunostaining for FAS, as opposed to 31.2 months, when staining was multifocal/diffuse (P = .005). Similar median survival values were obtained when cases were limited to endometrioid, serous, and mucinous carcinomas. Short-term survival at 1 and 2 years was significantly higher in patients whose tumors showed no or focal expression of FAS compared with multifocal/diffuse expression. Thus, elevated FAS may serve as an independent marker for predicting poor clinical outcome in patients with ovarian cancer.

Necrotizing cerebritis in an allogeneic bone marrow transplant recipient due to Cladophialophora bantiana.

We describe a necrotizing cerebritis in an allogeneic bone marrow transplant recipient caused by the neurotropic, dematiaceous fungus Cladophialophora bantiana. The patient presented 7 months after bone marrow transplantation with fever and sudden onset of left-sided weakness, followed shortly by cranial nerve III and VI palsies. The patient had a lesion (3.0 by 2.0 by 2.0 cm) of the right midbrain with extension to the pons, the left brain stem, and the right superior and the middle cerebellar peduncles. The diagnosis was made by microscopic examination and culture of a brain biopsy.

Fatty acid synthesis: a potential selective target for antineoplastic therapy.

OA-519 is a prognostic molecule found in tumor cells from breast cancer patients with markedly worsened prognosis. We purified OA-519 from human breast carcinoma cells, obtained its peptide sequence, and unambiguously identified it as fatty acid synthase through sequence homology and enzymology. Tumor fatty acid synthase is an approximately 270-kDa polypeptide which specifically abolished immunostaining of human breast cancers by anti-OA-519 antibodies. Tumor fatty acid synthase oxidized NADPH in a malonyl-CoA-dependent fashion and synthesized fatty acids composed of 80% palmitate, 10% myristate, and 10% stearate from acetyl-CoA, malonyl-CoA, and NADPH with a specific activity of 624 nmol of NADPH oxidized per min per mg. Tumor cell lines with elevated fatty acid synthase showed commensurate increases in incorporation of [U-14C]acetate into acylglycerols demonstrating that fatty acid synthase increases occur in the context of overall increases in endogenous fatty acid synthesis. Cerulenin inhibited acylglycerol synthesis in tumor cells and fibroblast controls in a dose-dependent fashion and also caused a growth inhibition which generally paralleled the level of endogenous fatty acid synthesis. Supraphysiologic levels of palmitate, 14 microM in dimethyl sulfoxide, significantly reversed the growth inhibition caused by cerulenin at concentrations of up to 5 micrograms/ml, indicating that cerulenin-mediated growth inhibition was due to fatty acid synthase inhibition.

Nephrogenic adenofibroma. A novel kidney tumor of young people.

Nephrogenic adenofibroma is a novel kidney tumor of young people (mean age of presentation, 13 years), who present with polycythemia, hypertension, or hematuria, which resolve following nephrectomy. The typical nephrectomy specimen contains a solitary, nonencapsulated, vaguely circumscribed, irregularly shaped or spherical, firm mass with either tan, gray-white, or pale yellow coloration. Cysts are sometimes present within the tumor. The histologic appearance is distinctive and characterized by a marked proliferation of spindled mesenchymal cells resembling the classical type of congenital mesoblastic nephroma, encasing discrete nodules of embryonal epithelium similar to the hyperplastic nephrogenic rests (nephroblastomatosis) usually associated with Wilms' tumor. The mesenchymal component consists of a fascicular proliferation of tightly interlaced, uniform, benign-appearing spindled cells that immunostatin for vimentin and fibronectin, but not desmin or actin. The epithelial component consists of discrete islands of blastemal cells that are partially or fully differentiated toward tubular, tubulopapillary, or papillary structures. Psammoma bodies are plentiful. Embryonal epithelium immunostains for cytokeratin but not epithelial membrane antigen. The overall histologic appearance of the mesenchymal and epithelial components is benign, and preliminary clinical data suggest that the tumor has a benevolent course. Two cases, however, contained small, well-circumscribed papillary lesions near the renal pelvis that resembled low-grade collecting duct carcinoma. The clinical implications of the latter finding are unclear.

Immunohistochemical localization of sodium-potassium-stimulated adenosine triphosphatase and carbonic anhydrase in human colon and colonic neoplasms.

Sodium-potassium-stimulated adenosine triphosphatase and carbonic anhydrase isozymes I and II were localized immunocytochemically in adenomas, adenocarcinomas, and normal epithelium of human colon harboring non-neoplastic lesions. Non-neoplastic control colon showed carbonic anhydrase I and II in the cytoplasm of the columnar cells lining the upper half of the crypts. Antiserum to sodium-potassium-stimulated adenosine triphosphatase bound to the basolateral but not the apical plasmalemma of columnar epithelial cells. Staining was most intense in the superficial cells, which also contained carbonic anhydrase, but was also evident to a lesser degree in cells deep in the crypts. Adenomas and adenocarcinomas failed to stain for content of carbonic anhydrase but retained basolateral sodium-potassium adenosine triphosphatase positivity. The staining characteristics of colonic neoplasms for the two enzymes involved in the transport function of colonic epithelium thus resembled those of the less mature cells lining the base of normal crypts.

Immunohistochemical localization of transport mediators in Wilms' tumor: comparison with fetal and mature human kidney.

Immunostaining for Na+, K+-ATPase, carbonic anhydrase (CA) II, and band 3 anion channel glycoprotein was compared in developing and mature human kidneys and in Wilms' tumors. In fetal kidneys, ATPase first appeared in proximal and distal tubules. At birth an adult pattern was present with abundant enzyme in all segments of the distal tubule and lesser amounts in proximal and collecting tubules. CA II was detected in fetal kidneys first in proximal and then in distal tubules and eventually, as in the adult, throughout the nephron. Band 3 glycoprotein was not detected in fetal kidneys and only weak staining was present in the basolateral plasmalemma of intercalated cells in newborn and infant kidneys. The number of cells reactive for band 3 and the intensity of staining in a given cell increased to near adult levels at about 2 years. This finding may provide a partial explanation for the 'physiological acidosis' characterized by a low systemic pH in newborn and young infants. ATPase was present in basolateral membranes of most epithelial cells in nonanaplastic Wilms' tumors but was absent in the epithelial component of two anaplastic Wilms' tumors. CA II was detected only in a few epithelial cells in four tumors. Neoplastic epithelial cells reactive for CA II also stained for ATPase but not vice versa. Band 3 glycoprotein was not detected in any Wilms' tumor. These findings show that the immunohistochemical assessment of protein involved in electrolyte transport provides a further means for determining the relative level of differentiation of tumor cells of epithelial origin and suggest that these methods may be a valuable aid in determining the prognosis of some carcinomas.

Beneficial effects of a 5-lipoxygenase inhibitor in endotoxic shock in the rat.

The effects of a highly selective 5-lipoxygenase inhibitor, CGS8515 [methyl 2-[(3,4-dihydro-3,4-dioxo-1-naphthalenyl) amino]benzoate], on endotoxic shock sequelae and eicosanoid synthesis by peritoneal macrophages were evaluated in the rat. Pretreatment of peritoneal macrophages in vitro with CGS8515 significantly inhibited the synthesis (P less than .01) of immunoreactive leukotriene C4/leukotriene D4 stimulated by the calcium ionophore (A23187). Inhibition of 5-lipoxygenase produced significant shunting to immunoreactive thromboxane B2 formation (P less than .05). In rats sedated with ketamine.HCl (82.5 mg/kg) and xylazine. HCl (27.5 mg/kg), i.v. injection of Salmonella enteritidis endotoxin (25 mg/kg i.v.) produced significant decreases at 30 min in mean arterial pressure (from 89 +/- 4 to 44 +/- 8 mm Hg, N = 5, P less than .001); in white blood cell count (from 10.8 +/- 0.6 to 6.5 +/- 0.8 x 10(3)/mm3, N = 5, P less than .01); in platelet count (from 687 +/- 66 to 392 +/- 65 x 10(3)/mm3, N = 5, P less than .01); and produced an increase of hematocrit (from 46 +/- 1.2 to 57.4 +/- 1.8%, N = 5, P less than .03). CGS8515 (5 mg/kg i.v. 30 min before endotoxin injection, N = 6) blunted the endotoxin-induced hypotension by 35% (P less than .001), the leukopenia by 24% (P less than .03), the thrombocytopenia by 45% (P less than .006) and the hemoconcentration by 16% (P less than .03), compared to the shocked control rats 30 min after endotoxin injection.(ABSTRACT TRUNCATED AT 250 WORDS)

The incidence and carbohydrate histochemistry of dystrophic goblet cells in colon.

A total of 512 colectomy and endoscopic biopsy specimens were reviewed to define the prevalence and possibly the significance of dystrophic goblet cells (DGCs) in neoplastic and nonneoplastic colonic diseases. As compared with an incidence of 1% in disease-free specimens, DGCs were observed in 38% of cases of inflammatory bowel disease, 23% of colonic malignancies, 30% of nonneoplastic polyps, 22% of adenomas, and 8% of cases showing acute self-limited colitis. In contrast, no dystrophic cells were seen in a group of miscellaneous diseases including diverticulitis, diverticulosis, abscesses, fistulas, ischemia, pseudomembranous colitis, melanosis coli, amyloidosis, shock, and mechanical trauma. Although dystrophic cells occur in association with dysplasia and carcinoma, their presence in nonpremalignant lesions, including acute self-limited colitis, raises doubt as to their diagnostic significance. Histochemical studies of the mucin composition in DGCs were unrevealing, failing to show any differences between DGCs and their morphologically normal counterparts in the same region of the colon.

Immunohistochemical localization of Na+,K+-ATPase in rodent and human salivary and lacrimal glands.

The enzyme Na+,K+-ATPase was localized immunohistochemically in major salivary glands of mouse, rat, and human and in exorbital lacrimal glands of the rodents. Immunoreactive Na+,K+-ATPase was abundant in the basolateral membranes of all epithelial cells lining striated and intra- and interlobular ducts of all glands. Reactivity of intercalated ducts varied among gland type and species. Cells lining granular ducts in rodent submandibular gland showed a heterogeneous staining pattern in rat but stained homogeneously in mouse. Secretory cells varied greatly in their content of immunoreactive Na+,K+-ATPase. As with all duct cells, staining was present only at the basolateral surface and was never observed at the luminal surface of reactive secretory cells. Mucous cells failed to show any reactivity in any gland examined. Serous cells showed a gradient of immunostaining intensity ranging from strongly positive in demilunes of human sublingual gland to negative in rat submandibular gland and lacrimal glands of rats and mice. The presence of basolaterally localized Na+,K+-ATPase in most serous cells but not in mucous cells suggests that the enzyme contributes to the ion and water content of copious, low-protein serous secretions. The intense immunostaining of cells in most if not all segments of the duct system supports the idea that the ducts are involved with modification of the primary saliva, and extends this concept to include all segments of the duct system.

Distribution of fucosubstance in kidney and related neoplasms. Absence of lectin-reactive alpha-fucose from the vasculature of bilateral Wilm's tumors.

Two alpha-fucose-binding lectins, Ulex europaeus agglutinin I (UEA I) and Lotus tetragonolobus agglutinin, were employed to compare and contrast the distribution of fucosubstance in normal human kidneys and a variety of renal tumors. The study employed a total of 31 kidneys surgically removed for the presence of a variety of tumors, including 11 unilateral Wilms' tumors, two cases of bilateral Wilms' tumors, 13 renal cell carcinomas, two congenital mesoblastic nephromas, one renal oncocytoma, one neuroblastoma metastatic to the kidney, and one clear cell sarcoma of the kidney. The results show that UEA I-reactive fucosubstance is detected in vascular endothelium of all kidneys and tumors, except bilateral Wilms' tumors. The presence of UEA I-reactive alpha-fucose in the vasculature of unilateral but not bilateral Wilms' tumors defines a unique histochemical distinction between the two groups of tumors. Conceivably, this property might be exploited as a screening procedure for the more aggressive bilateral neoplasms. Other findings detail histochemical differences between UEA I and L tetragonolobus agglutinin, as evidenced by the ability of one lectin to stain a particular cell type that is not reactive with the other lectin.

Glomerular sialoconjugates of developing and mature rat kidneys.

The appearance of sialoconjugates in developing rat kidney glomeruli was studied using lectins and neuraminidase-lectin staining sequences. In the early S-shaped bodies, binding of Maclura pomifera (MPA; specific for galactosaminyl residues of glycoconjugates) could be detected in the presumptive podocyte layer at the apex of these cells, but notably no binding of lectins specific for sialic acid could be seen. During further morphologic maturation of the S-shaped bodies, binding of Limax flavus (LFA; specific for sialic acids) and Triticum vulgaris (WGA; specific for sialic acids and N-acetyl glucosaminyl moieties) appeared at the apex of podocytes and extended subsequently along the lateral membranes to the base of these cells. In morphologically mature glomeruli, LFA stained not only the base of podocytes but also glomerular basement membranes. WGA and MPA bound to the capillary endothelia as well as to the structures bound by LFA. The intensity of WGA binding increased considerably after 5 days of postnatal life, seemingly in parallel with the decrease and ultimate disappearance of MPA binding. In addition to showing individual appearance pattern for various lectin binding sites, these studies give evidence of previously unrecognized postnatal completion of the components of glomerular filtration barrier.

Genetic differences in the histochemically defined structure of oligosaccharides in mice.

A wide range of tissues from three interfertile species of mice and an interspecific hybrid was examined with lectins conjugated to peroxidase to localize specifically glycoconjugates containing terminal alpha-N-acetylgalactosamine, alpha-galactose, and alpha-fucose, and the terminal disaccharide galactose-(beta 1----3)-N-acetylgalactosamine. This battery of lectins disclosed marked heterogeneity of glycoconjugates in different histological sites in a given animal and even between cells in a presumably homogeneous cell population within an organ. No variation with any lectin was observed between individuals of two closely related inbred strains of Mus domesticus at any specific histological or cytological site. In contrast, littermates of an outbred strain of Mus castaneus differed in binding of certain lectins at various sites, attesting to a genetic basis for individual variation. Hybrids between castaneus and domesticus mice also showed individual variation. Moreover, extensive differences between the mouse species were demonstrable with every lectin in glycoconjugates of stored secretions, Golgi cisternae, and apical or basolateral plasmalemma in many cell types. Totaling the differences in tabulated staining intensities for each possible species pair gave a measure of the overall extent of difference at 53 histological sites. According to this measure, the three species are about equally divergent from one another. Some differences between species appeared to depend on histological rather than histochemical variation, as, for example, a greater abundance of granular duct cells in the sublingual and submandibular glands in Mus hortulanus. Other differences were apparently derived from pathological change, as exemplified by casts and lymphoid infiltrates in kidney and structurally atypical submandibular gland lobules in Mus castaneus, and possibly by infiltrating cells in intestinal lamina propria and epithelium in Mus castaneus and hortulanus.

Lectin detection of renal glycogen in rats with short-term streptozotocin-diabetes.

Earlier histochemical findings from our laboratory have shown that a lectin (agglutinin) from Griffonia simplicifolia, which reportedly binds to terminal N-acetylglucosamine residues in glycoconjugate oligosaccharides also shows affinity for glycogen. In the present study, the lectin was conjugated to horseradish peroxidase and applied to paraffin sections of kidney from streptozotocin-diabetic rats, insulin-treated and untreated, and age-matched control rats. Griffonia simplicifolia agglutinin II detected glycogen in cortical ascending thick limbs of untreated diabetic rat kidneys as early as 24 h following injection of streptozotocin. The number of stained cells increased steadily so that by day 14 of diabetes the lectin reacted with nearly all of the cells lining ascending thick limbs in the cortex and adjacent outer stripe of the outer medulla. Glycogen was never identified in the inner medullary stripe. Comparison of Griffonia simplicifolia agglutinin II and periodic acid-Schiff staining revealed that periodic acid-Schiff could not clearly detect glycogen until 14 days following injection of streptozotocin, which substantiated earlier claims that Griffonia simplicifolia agglutinin II might be a more sensitive indicator of glycogen than periodic acid-Schiff. The distribution of glycoconjugate containing terminal N-acetylglucosamine stainable with the lectin was unchanged in diabetic kidneys. Griffonia simplicifolia agglutinin II served in the present study to further characterise the sequence of abnormal glycogen accumulation in streptozotocin-diabetic rat kidneys. In addition, it was shown that the lectin's ability to antedate periodic acid-Schiff detection of glycogen has utility in histochemical investigations in diabetes.

Histochemical specificity of beta-galactose binding lectins from Arachis hypogaea (peanut) and Ricinus communis (castor bean).

Previous histochemical studies have demonstrated disparities in the binding of two lectins with a nominal specificity for terminal beta-D-galactose. Biochemical studies have shown that the most complementary structure for binding peanut agglutinin (PNA) is the terminal disaccharide Gal-(beta 1----3)-GalNAc, whereas the most complementary structure for binding Ricinus communis agglutinin I (RCA I) is the terminal disaccharide Gal-(beta 1----4)-GlcNAc. However, it is not known if only these differences in affinity account for the different histochemical staining reactions observed on tissue sections. In the present study we compared the staining patterns of PNA and RCA I by inhibiting in situ the binding of each lectin conjugated to horseradish peroxidase (HRP) with increasing concentrations of unlabeled PNA or RCA I. The PNA-HRP conjugate did not stain most tissue sites suspected of containing an abundance of glycoconjugates with terminal Gal-(beta 1----4)-GlcNAc. Moreover, unlabeled PNA failed to significantly inhibit strong RCA I-HRP staining in these sites. In loci thought to contain variable amounts of glycoconjugates with terminal Gal-(beta 1----3)-GalNAc, unlabeled RCA I decreased PNA-HRP reactivity only slightly or not at all, whereas weak to strong RCA I-HRP staining was diminished or abolished by unlabeled PNA. The results suggest that PNA staining is restricted to glycoconjugates with terminal Gal-(beta 1----3)-GalNAc. RCA I apparently reacts most strongly with glycoconjugates having the terminal disaccharide Gal-(beta 1----4)-GlcNAc, but also stains sites containing a moderate to abundant amount of glycoconjugates with the terminal Gal-(beta 1----3)-GalNAc sequence.