PMS2: a potential prognostic protein marker in oral squamous cell carcinoma.

BACKGROUND: An increase in oral squamous cell carcinoma (OSCC) cases was observed despite the reduction in exposure to classic risk factors. Although the exact cause of this trend remains unknown, epigenetic factors could be contributing to an increased occurrence of these tumors. This study aims to assess the influence of PMS2 protein immunoexpression on the prognosis of patients with OSCC. MATERIAL AND METHODS: This study comprised 76 cases of OSCC treated between 2011 and 2016. Immunohistochemical staining for PMS2 was performed. For evaluation, 10 fields per histological section were photographed at a 400x magnification and positively-stained cells were counted with Image J. Mann-Whitney and Kruskal-Wallis tests were used to compare the immunolabeling pattern with the clinical-pathological and prognostic characteristics. Survival analysis was performed with Chi-square, Long-Rank Mantel-Cox and Cox regression tests (p<0.05). RESULTS: An overexpression of PMS2 was observed in N0/1 tumors and in oral cancers found in unusual locations. In patients ≤60 years of age, high levels of PMS2 (>60%; p=0.041) were associated with low survival (p=0.029). In multivariate analysis, surgery combined with chemotherapy (p=0.030) and high PMS2 immunoexpression (p=0.042) significantly increased the risk of death for ≤60 years old patients. CONCLUSIONS: The findings of this study indicate that PMS2 can be a potential prognostic protein marker in OSCC patients 60 years of age and younger.

C-type natriuretic peptide modulates bidirectional plasticity in hippocampal area CA1 in vitro.

C-type natriuretic peptide (CNP) and the natriuretic peptide receptor B (NPR-B) are expressed throughout the hippocampus. We tested whether CNP affected long-term potentiation (LTP) or long-term depression (LTD) in area CA1. Field potentials (FP) were simultaneously recorded in stratum pyramidale (SP) and stratum radiatum (SR) of area CA1 in rat hippocampal slices. To induce LTD and LTP stimulation was applied to SR in area CA1 at 1 and 5 Hz and 30-100 Hz, respectively. CNP (100 nM) increased LTD magnitude while LTP induction was impeded. Thus, in the presence of CNP the threshold for LTP induction was shifted to higher stimulus frequencies, a modulation that showed layer-specific differences in area CA1. Effects of CNP were prevented by the NPR-B antagonist HS-142-1. In the presence of the GABA(A) receptor blocker bicuculline (BMI, 5 microM), CNP-mediated effects were attenuated in SP and SR. Intracellular recordings under this condition revealed that CNP significantly reduced number of action potentials generated during depolarizing current steps. The input resistance of CA1 cells and amplitude of isolated excitatory postsynaptic potential (EPSPs) were significantly increased by CNP whereas these changes were not observed in the absence of BMI. 100 Hz stimulation induced stable potentiation of the EPSP amplitude in CA1 pyramidal cells while this effect was strongly attenuated by CNP. This effect was prevented by BMI. Immunohistochemistry indicated that the peptide binds to receptors expressed on pyramidal cells and GAD(65/67)-immunopositive interneurons. 20 Hz stimulation, applied for 30 s, induced LTP in SR and SP. CNP attenuated LTP in SP and reversed LTP into LTD in SR. These effects were mimicked by low-dose dl-2-amino-5-phosphonopentanoic acid (dl-APV) (10 microM) suggesting partial N-methyl d-aspartate (NMDA) receptor dependency of CNP-mediated effects. Together, our data suggest that CNP is involved in the regulation of bidirectional plasticity in area CA1 potentially by modulating GABA(A)-mediated inhibition and NMDA receptors.

C-type natriuretic peptide decreases hippocampal network oscillations in adult rats in vitro.

C-type natriuretic peptide (CNP) is an abundant neuropeptide in the human brain and the cerebrospinal fluid. CNP is involved in anxiogenesis and exerts its effects through the natriuretic peptide receptor B (NPR-B), which is expressed in the hippocampus. Hippocampal network oscillations of distinct frequency bands like gamma (gamma)-oscillations and sharp wave-ripple complexes (SPW-Rs) are likely involved in various cognitive functions such as the storage of information and memory consolidation in vivo. Here, we tested the effects of CNP on distinct network oscillations in horizontal slices of rat hippocampus. We found that CNP decreased the power of stimulus- and ACh/physostigmine-induced gamma-oscillations. In contrast to stimulus-induced gamma-oscillations, CNP increased the frequency of ACh-induced, persistent network oscillations. Moreover, the peptide hormone reduced the incidence of LTP-associated SPW-Rs in area CA3 and CA1. Immunohistochemistry indicates that the peptide binds to receptors expressed on a subset of GAD 65-67-immunopositive cells in addition to binding to principal and other presumably non-neuronal cells. CNP caused a hyperpolarization of CA3 neurons increased their input resistance and decreased inhibitory conductance. Together, our data suggest that the effects of CNP on synchronized hippocampal network oscillations might involve effects on hippocampal interneurons.

Severe gastrointestinal disease due to HIV-1-seronegative AIDS.

An HIV-1 seronegative man presented with odynophagia, dysphagia, diarrhea, tenesmus and a 50-lb weight loss. A large esophageal ulcer and a rectal fissure were identified endoscopically. Stool samples and biopsy specimens from the esophageal ulcer, duodenum, colon and rectum were negative for pathogens. Seronegative AIDS was suspected, and high levels of HIV-1 mRNA (> 242,000 copies/mL) were detected. The esophageal ulcer responded to oral steroids and the HIV-1 infection to highly active anti-retroviral therapy (HAART). The virus isolated from the patient and an HIV-1 seropositive, asymptomatic, female sex worker with whom he had recently terminated a one-year heterosexual relationship showed sequence homology, indicating her as the source of his virus. The unusual presentation of severe gastrointestinal disease in an HIV-1 seronegative man with HIV-1 viremia underscores the importance of including AIDS in the differential diagnosis of wasting syndrome (i. e., B-type symptoms such as fever, night sweats, weight loss) in patients who are HIV-1 seronegative but at risk for AIDS.

Neutralizing antibody responses in acute human immunodeficiency virus type 1 subtype C infection.

The study of the evolution and specificities of neutralizing antibodies during the course of human immunodeficiency virus type 1 (HIV-1) infection may be important in the discovery of possible targets for vaccine design. In this study, we assessed the autologous and heterologous neutralization responses of 14 HIV-1 subtype C-infected individuals, using envelope clones obtained within the first 2 months postinfection. Our data show that potent but relatively strain-specific neutralizing antibodies develop within 3 to 12 months of HIV-1 infection. The magnitude of this response was associated with shorter V1-to-V5 envelope lengths and fewer glycosylation sites, particularly in the V1-V2 region. Anti-MPER antibodies were detected in 4 of 14 individuals within a year of infection, while antibodies to CD4-induced (CD4i) epitopes developed to high titers in 12 participants, in most cases before the development of autologous neutralizing antibodies. However, neither anti-MPER nor anti-CD4i antibody specificity conferred neutralization breadth. These data provide insights into the kinetics, potency, breadth, and epitope specificity of neutralizing antibody responses in acute HIV-1 subtype C infection.

Sensitivity of human immunodeficiency virus type 1 to fusion inhibitors targeted to the gp41 first heptad repeat involves distinct regions of gp41 and is consistently modulated by gp120 interactions with the coreceptor.

T-20 is a synthetic peptide that corresponds to 36 amino acids within the C-terminal heptad repeat region (HR2) of human immunodeficiency virus type 1 (HIV-1) gp41. T-20 has been shown to potently inhibit viral replication of HIV-1 both in vitro and in vivo and is currently being evaluated in a Phase III clinical trial. T-649 is an inhibitory peptide that also corresponds to 36 amino acids within HR2. This sequence overlaps the T-20 sequence but is shifted 10 residues toward the N terminus of gp41. Both inhibitors are thought to exert their antiviral activity by interfering with the conformational changes that occur within gp41 to promote membrane fusion following gp120 interactions with CD4 and coreceptor molecules. We have shown previously that coreceptor specificity defined by the V3 loop of gp120 modulates sensitivity to T-20 and that a critical region within the N-terminal heptad repeat (HR1) of gp41 is the major determinant of sensitivity (C. A. Derdeyn et al., J. Virol. 74:8358-8367, 2000). This report shows that (i) regions within gp41 distinct from those associated with T-20 sensitivity govern the baseline sensitivity to T-649 and (ii) T-649 sensitivity of chimeric viruses that contain sequences derived from CXCR4- and CCR5-specific envelopes is also modulated by coreceptor specificity. Moreover, the pattern of sensitivity of CCR5-specific chimeras with only minor differences in their V3 loop was consistent for both inhibitors, suggesting that the individual affinity for coreceptor may influence accessibility of these inhibitors to their target sequence. Finally, an analysis of the sensitivity of 55 primary, inhibitor-naive HIV-1 isolates found that higher concentrations of T-20 (P < 0.001) and T-649 (P = 0.016) were required to inhibit CCR5-specific viruses compared to viruses that utilize CXCR4. The results presented here implicate gp120-coreceptor interactions in driving the complex conformational changes that occur in gp41 to promote fusion and entry and suggest that sensitivity to different HR1-directed fusion inhibitors is governed by distinct regions of gp41 but is consistently modulated by coreceptor specificity.

Teaching resuscitation to pediatric residents: the effects of an intervention.

OBJECTIVE: To evaluate the effectiveness of an educational intervention on pediatric residents' resuscitation fund of knowledge, technical skills, confidence, and overall performance. DESIGN: Prospective, nonconcurrent, controlled interventional trial. SETTING: Urban pediatric tertiary care hospital. PARTICIPANTS: An intervention group (IG) of 28 pediatric residents graduating in 1997, and a control group (CG) of 30 pediatric residents graduating in 1996. INTERVENTIONS: Resuscitation course with didactic lectures and skills practice stations, as well as a minimum of 3 practice mock resuscitations with immediate feedback throughout postgraduate year 3. MAIN OUTCOME MEASURES: Fund of knowledge, using the Pediatric Advanced Life Support test and short answer test; technical skills, using the Airway and Vascular Access Skills Assessment; experience and confidence, using an anonymous survey; and overall performance, evaluated using a videotaped mock resuscitation test. RESULTS: The IG scored better on the short answer test (P<.001). A larger number of IG residents were successful in the completion of ancillary airway maneuvers and femoral vascular access (P =.02), as well as endotracheal intubation (P =.004) and intraosseous access (P =.002). The IG was more confident in their leadership role (P =.0001) and technical skills (P =.05). Trends toward improved overall performance were noted for the IG mock resuscitations. Residents in the IG were more likely to assess the airway in fewer than 2 minutes (P =.02), recognize the threat to life in fewer than 5 minutes (P =.02), and complete the primary survey in a timely fashion (P =.05). They required fewer prompts (P =.04) and made fewer mistakes (P =.07). CONCLUSIONS: A structured, formal curriculum can improve the necessary fund of knowledge, skills, confidence, and leadership required for resuscitation.

Sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by coreceptor specificity defined by the V3 loop of gp120.

T-20 is a synthetic peptide that potently inhibits replication of human immunodeficiency virus type 1 by interfering with the transition of the transmembrane protein, gp41, to a fusion active state following interactions of the surface glycoprotein, gp120, with CD4 and coreceptor molecules displayed on the target cell surface. Although T-20 is postulated to interact with an N-terminal heptad repeat within gp41 in a trans-dominant manner, we show here that sensitivity to T-20 is strongly influenced by coreceptor specificity. When 14 T-20-naive primary isolates were analyzed for sensitivity to T-20, the mean 50% inhibitory concentration (IC(50)) for isolates that utilize CCR5 for entry (R5 viruses) was 0.8 log(10) higher than the mean IC(50) for CXCR4 (X4) isolates (P = 0. 0055). Using NL4.3-based envelope chimeras that contain combinations of envelope sequences derived from R5 and X4 viruses, we found that determinants of coreceptor specificity contained within the gp120 V3 loop modulate this sensitivity to T-20. The IC(50) for all chimeric envelope viruses containing R5 V3 sequences was 0.6 to 0.8 log(10) higher than that for viruses containing X4 V3 sequences. In addition, we confirmed that the N-terminal heptad repeat of gp41 determines the baseline sensitivity to T-20 and that the IC(50) for viruses containing GIV at amino acid residues 36 to 38 was 1.0 log(10) lower than the IC(50) for viruses containing a G-to-D substitution. The results of this study show that gp120-coreceptor interactions and the gp41 N-terminal heptad repeat independently contribute to sensitivity to T-20. These results have important implications for the therapeutic uses of T-20 as well as for unraveling the complex mechanisms of virus fusion and entry.

Assessing pediatric senior residents' training in resuscitation: fund of knowledge, technical skills, and perception of confidence.

OBJECTIVE: To describe pediatric housestaff knowledge, experience, confidence in pediatric resuscitations and their ability to perform important resuscitation procedures during the usual training experience. DESIGN AND PARTICIPANTS: Cohort study of PGY-3 level residents in a ACGME accredited pediatric residency training program at a large, tertiary care children's hospital. METHODS: Fund of knowledge was assessed by administering the standardized test from the Pediatric Advanced Life Support (PALS) Course in addition to a supplemental short answer test requiring clinical problem-solving skills. Procedural skills were evaluated through observation of the resident performing four procedures during a skills workshop using a weighted step-wise grading sheet. Resident experience and confidence was quantified using an anonymous survey. RESULTS: Ninety-seven percent of residents participated. Residents achieved high scores on the standardized PALS test (93.2%+/-5.5), but performed less well when answering more complicated questions (60.0%+/-9.9) on the short answer test. No resident was able to successfully perform both basic and advanced airway skills, and only 11% successfully completed both vascular skills. Although residents were overall confident in their resuscitation skills, performance in the skill workshop revealed significant deficits. For example, only 18% performed ancillary airway maneuvers properly. None of the residents performed all four skills correctly. Experience in both real and mock resuscitations was infrequent. Residents reported receiving feedback on their performance less than half of the time. Over 89% of them felt that resuscitation knowledge and skill were important for their future chosen career. CONCLUSION: Pediatric residents infrequently lead or participate in real or mock resuscitations. Although confident in performing many of the necessary resuscitation skills, few residents performed critical components of these skills correctly. Current pediatric residency training may not provide sufficient experience to develop adequate skills, fund of knowledge, or confidence needed for resuscitation.

The immune system: relation to sepsis and multiple organ failure.

The immune system plays a dual role in the pathogenesis of sepsis and organ failure, intended for host defense but also possessing significant cytodestructive capacity. As the understanding of the epidemiology and pathophysiology of these disorders improves, so too does the appreciation for the complexity of this system. No longer is the immune response viewed as simply cellular or humoral but rather as a network of cells, chemical mediators, and molecular elements. The interactions between these various components serve to regulate and coordinate the inflammatory response. When this fine balance is lost, the inflammatory response becomes pathologic and self-destructive. Organ injury ensues, and with this injury, further escalation of the inflammatory response occurs; becoming a self-perpetuating process. Conventional therapy is limited to supportive care and has been ineffective in improving mortality. To date, efforts to modulate the inflammatory response by inhibition of specific components have been unsuccessful. In the future, better patient selection, combination therapy (perhaps using strategies of early augmentation followed by inhibition), and alternative techniques such as blood purification may prove to be more effective.

Nebulized dexamethasone versus oral prednisone in the emergency treatment of asthmatic children.

STUDY OBJECTIVE: To compare nebulized dexamethasone with oral prednisone in the treatment of children with asthma. DESIGN: A randomized, double-blind, double-placebo study. SETTING: An urban pediatric emergency department. PARTICIPANTS: Patients aged 1 to 17 years with acute asthma. INTERVENTIONS: Patients with moderate asthma exacerbation received frequent aerosolized albuterol and either 1.5 mg/kg of nebulized dexamethasone or 2 mg/kg of oral prednisone. RESULTS: A total of 111 children was evaluated; 21% of those treated with dexamethasone required hospitalization, compared with 31% of those treated with prednisone (P = .26). A significantly greater proportion of dexamethasone-treated children were discharged home within 2 hours (23% versus 7%, P = .02). In the dexamethasone group, 8% who received the drug by mouthpiece were hospitalized compared with 33% who received it by face mask (P = .06). Fewer children treated with dexamethasone vomited (0% versus 15%, P = .001) and fewer relapsed within 48 hours of ED discharge (0% versus 16%, P = .008). CONCLUSION: Nebulized dexamethasone was as effective as oral prednisone in the ED treatment of moderately ill children with acute asthma and was associated with more rapid clinical improvement, more reliable drug delivery, and fewer relapses.

Activity of triciribine and triciribine-5'-monophosphate against human immunodeficiency virus types 1 and 2.

Triciribine (TCN) and its 5'-monophosphate (TCN-P) are novel tricyclic compounds with known antitumor activity; TCN-P is currently in phase II human clinical trials. We now report that these compounds have potent and selective activity against HIV-1 and HIV-2. Using a syncytial plaque assay, TCN and TCN-P were active against HIV-1 at 0.01-0.02 microM and had differential selectivities of 2250 and 1900, respectively, compared to 1850 for AZT. In contrast, TCN and TCN-P had minimal selectivity against human cytomegalovirus (50 and 27, respectively). TCN and TCN-P markedly inhibited HIV-1-induced p24 core antigen production, reverse transcriptase, and infectious virus production in a dose-dependent manner using HIV-1 acutely infected CEM-SS, H9, and persistently infected H9IIIB and U1 cells. In acutely infected PBL cells, TCN and TCN-P inhibited reverse transcriptase and infectious virus production but not p24 core antigen production. Using a microtiter XTT assay, TCN and TCN-P were active against a panel of HIV-1 and HIV-2 strains at IC50 values ranging from 0.02 to 0.46 microM. Evaluation of matched pairs of predrug and postdrug therapy HIV-1 isolates established that AZT-resistant and TIBO-resistant variants of HIV-1 were sensitive to TCN or TCN-P. Furthermore, unlike AZT and other fraudulent nucleosides, neither TCN, TCN-P, nor TCN-TP inhibited the viral reverse transcriptase. Thus, even though triciribine is a nucleoside chemically, it does not act biologically by classic nucleoside modalities but rather by a unique mechanism yet to be elucidated.

Evidence that specific high mannose structures directly regulate multiple cellular activities.

Previous studies have demonstrated that much of the immunomodulatory activity of the glycoprotein uromodulin can be attributed to attached oligosaccharides. Structural studies of isolated and purified saccharides derived from uromodulin suggest that the structure Man6GlcNAc2-asn can inhibit in vitro assays of antigen driven T cell proliferation. Based on these observations, we isolated a series of high mannose glycopeptides from a variety of natural sources and tested them for biological activity in a number of assays. We found that purified mannose rich glycopeptides are able to activate the hexose monophosphate (HMP) shunt, induce prostaglandin synthesis, and directly stimulate IL-1 synthesis. These in vitro effects appear to have in vivo counterparts. Thus in a species-restricted fashion, high mannose compounds are able to directly activate a delayed mononuclear cell infiltrate after intradermal injection. Our data suggest that specific mannose oligosaccharides may activate as well as inhibit cellular immune responses at several different levels. These findings support the hypothesis that specific saccharide structures could participate in the physiologic regulation of the immune response.

Uromodulin: a specific inhibitor of IL-1-initiated human T cell colony formation.

Uromodulin, an 85 kDa naturally occurring immunosuppressant, was found to selectively and specifically inhibit the ability of IL-1 to induce colony responses by highly enriched suspensions of PHA-stimulated T lymphocytes. Dilutions of 1 x 10(-8) M completely blocked the colony growth of T lymphocytes cultured with 50 U/ml IL-1; 1 x 10(-9) M dilutions reduced scores by 83%. By contrast, uromodulin did not inhibit the responses of unseparated mononuclear cells, isolated T lymphocytes cultured with irradiated adherent cells, or stimulated T cells whose growth was initiated by either IL-2 or a soluble factor derived from Raji cells.

Pregnancy-associated changes in oligomannose oligosaccharides of human and bovine uromodulin (Tamm-Horsfall glycoprotein).

The urinary glycoprotein uromodulin (Tamm-Horsfall glycoprotein) exhibits a pregnancy-associated ability to inhibit antigen-specific T cell proliferation, and the activity is associated with a carbohydrate moiety [Muchmore and Decker (1985) Science 229:479-81; Hession et al., (1987) Science 237:1479-84; Muchmore, Shifrin and Decker (1987) J Immunol 138:2547-53]. We report here that the Man6(7)GlcNAc2-R glycopeptides derived from uromodulin inhibit antigen-specific T cell proliferation by 50% at 0.2-2 microM, and further studies, reported elsewhere, confirm that oligomannose glycopeptides from other sources are also inhibitory, with Man9GlcNAc2-R the most inhibitory of those tested [Muchmore et al., J Leukocyte Biol (in press)]. In this work, we have extended the observation of pregnancy-associated inhibitory activity to a second species, and have compared the oligomannose profile of Tamm-Horsfall glycoprotein (nonpregnant) with that of uromodulin (pregnant) derived from both human and bovine sources. Surprisingly, there was a pregnancy-associated decrease in the total content of oligomannose chains due predominantly to a reduction in Man5GlcNAc2-R and Man6GlcNAc2-R. Man7GlcNAc2-R, which did not decrease with pregnancy, comprised a significantly greater proportion of the total oligomannose chains in pregnant vs. nonpregnant samples from both species (human; 34.6% vs. 25.9%: bovine; 14.4% vs. 7.2%).

IL-2, a lectin with specificity for high mannose glycopeptides.

Utilizing a solid phase binding assay, we have demonstrated that rIL-2 binds with high affinity to the human urinary glycoprotein uromodulin. This binding is specifically inhibited by the saccharides diacetylchitobiose and Man(alpha 1-3)(Man(alpha 1-6]Man-O-methyl and by the high mannose glycopeptides Man5GlcNAc2-R and Man6GlcNAc2-R, but not by Man9GlcNAc2-R. rIL-2 also binds OVA, a glycoprotein which contains approximately 50% high mannose chains at a single glycosylation site, and to yeast mannan. This binding is inhibited by the same battery of saccharides which inhibit the binding to uromodulin. The conclusion that rIL-2 is a lectin is further supported by the observation that the sequence of IL-2 shares 27% homology with a 33-residue sequence of the carbohydrate-binding domain of human mannose-binding protein. The potential physiologic relevance of the carbohydrate binding activity is further elucidated by studies which show that 1) binding of soluble rIL-2 to immobilized uromodulin is enhanced at a pH of 4 to5 in the presence of divalent cations, and 2) neither uromodulin nor the high mannose glycopeptide Man5GlcNAc2Asn blocks the binding of rIL-2 to the IL-2R. Thus the carbohydrate-binding site of rIL-2 is distinct from the cell surface receptor-binding site, and might function preferentially in acidic microenvironments.

The lectin-like interaction between recombinant tumor necrosis factor and uromodulin.

The polypeptide of uromodulin, an immunosuppressive glycoprotein isolated from human urine, has been shown to be identical to that of Tamm-Horsfall glycoprotein and is synthesized exclusively in the kidney (Hession, C., Decker, J. M., Sherblom, A. P., Kumar, S. (1987) Science 237, 1479-1484). Uromodulin binds recombinant murine interleukin 1 alpha with high affinity, and this binding can be inhibited by addition of specific saccharides (Muchmore, A. V., and Decker, J. M. (1987) J. Immunol. 138, 2541-2546). We now report that uromodulin binds recombinant human tumor necrosis factor (rTNF) with high affinity. Both diacetylchitobiose and Man(alpha 1-6)(Man(alpha 1-3]-Man-O-ethyl are effective inhibitors of the binding, whereas a wide variety of other saccharides are not inhibitory. Although Tamm-Horsfall glycoprotein contains predominantly tetraantennary N-linked chains, the binding to rTNF is unaffected by removal of terminal sialic acid, galactose, and N-acetylhexosamine residues. Fractionation of a Pronase digest of uromodulin by gel filtration yields material that inhibits the binding of uromodulin to rTNF but is of lower molecular weight than the major oligosaccharide. Uromodulin does not inhibit the cytotoxic activity of rTNF as monitored by lysis of tumor cell targets but effectively protects mice from lethal challenge with lipopolysaccharide, an event that may involve lymphokine toxicity. We have previously shown that rTNF binds to sections of human kidney and is localized in the same region as uromodulin. Thus, rTNF interacts with uromodulin via carbohydrate chains that are less processed than the major tetraantennary chain, and this interaction may be critical in promoting clearance and/or reducing toxicity of TNF and other lymphokines.

Uromodulin (Tamm-Horsfall glycoprotein): a renal ligand for lymphokines.

The protein portion of the immunosuppressive glycoprotein uromodulin is identical to the Tamm-Horsfall urinary glycoprotein and is synthesized in the kidney. Evidence that the glycoproteins are the same is based on amino acid sequence identity, immunologic cross-reactivity, and tissue localization to the thick ascending limb of Henle's loop. Nucleic acid sequencing of clones for uromodulin isolated from a complementary DNA bank from human kidney predicts a protein 639 amino acids in length, including a 24--amino acid leader sequence and a cysteine-rich mature protein with eight potential glycosylation sites. Uromodulin and preparations of Tamm-Horsfall glycoprotein bind to recombinant murine interleukin-1 (rIL-1) and human rIL-1 alpha, rIL-1 beta, and recombinant tumor necrosis factor (rTNF). Uromodulin isolated from urine of pregnant women by lectin adherence is more immunosuppressive than material isolated by the original salt-precipitation protocol of Tamm and Horsfall. Immunohistologic studies demonstrate that rIL-1 and rTNF bind to the same area of the human kidney that binds to antiserum specific for uromodulin. Thus, uromodulin (Tamm-Horsfall glycoprotein) may function as a unique renal regulatory glycoprotein that specifically binds to and regulates the circulating activity of a number of potent cytokines, including IL-1 and TNF.

Evidence that recombinant IL 1 alpha exhibits lectin-like specificity and binds to homogeneous uromodulin via N-linked oligosaccharides.

Uromodulin, a recently described immunosuppressive glycoprotein isolated from human pregnancy urine, has been shown to inhibit T cell proliferative assays dependent upon interleukin 1 (IL 1). We have also recently demonstrated that uromodulin binds specifically to IL 1. We now show that not only the biologic activity but also the binding affinity of uromodulin for recombinant IL 1 is dependent upon intact glycosylation. Furthermore, oligosaccharides isolated from pronase-digested uromodulin are immunosuppressive by themselves and are able to compete with native uromodulin for binding to IL 1. We conclude that recombinant IL 1 exhibits lectin-like specificity, and uromodulin is a biologically functional glycoprotein target of the lectin-like specificity of IL 1.

In vitro evidence that carbohydrate moieties derived from uromodulin, an 85,000 dalton immunosuppressive glycoprotein isolated from human pregnancy urine, are immunosuppressive in the absence of intact protein.

Our laboratory recently reported the purification of a unique immunosuppressive glycoprotein isolated from human pregnancy urine (7). This glycoprotein, which we term uromodulin, has a m.w. of 85,000 as assessed on SDS-PAGE and is 30% carbohydrate. Uromodulin blocks in vitro antigen-specific T cell proliferation to recall antigens such as tetanus toxoid at concentrations as low as 100 pM. This glycoprotein also blocks the in vitro generation of spontaneous monocyte-mediated cytotoxicity (7, 36). Recent evidence strongly suggests that the primary action of uromodulin is to act as a specific ligand and modulator of IL 1 (10, 33). We now report additional biochemical characterization of uromodulin, and based on three independent lines of evidence, find that its immunologic activity appears to result from its glycosylation. First, measures to alter the tertiary folding of the protein backbone of uromodulin, including succinylation or reduction and carboxymethylation, fail to significantly affect its in vitro bioactivity. Second, after extensive digestion of intact uromodulin with pronase, the majority of the in vitro bioactivity can be recovered in a single carbohydrate-rich fraction. Finally, digestion with N-glycanase (N-glycosidase F-, an enzyme specific for N-asparagine-linked oligosaccharides) and subsequent purification on thin layer chromatography yields a single complex oligosaccharide that appears to be responsible for the majority of the in vitro immunosuppression mediated by uromodulin. These data suggest that uromodulin displays N-linked carbohydrate sequences capable of down-regulating antigen-specific T cell responses in vitro. It has been suggested that endogenous lectins may play an important role as recognition molecules in mammalian, as well as more primitive immune systems (23, 24). Our in vitro biologic data strongly suggest that the carbohydrate portion of uromodulin is an excellent candidate to function as a potential lectin receptor.