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Antonietta M Lillo,1 Nileena Velappan,1 Julia M Kelliher,1 Austin J Watts,1 Samuel P Merriman,1 Grace Vuyisich,2 Laura M Lilley,2 Kent E Coombs,1 Tara Mastren,2 Munehiro Teshima,1 Benjamin W Stein,2 Gregory L Wagner,2 Srinivas Iyer,1 Andrew RM Bradbury,3 Jennifer Foster Harris,1 Armand E Dichosa,1 Stosh A Kozimor2 1Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA; 2Chemistry Division, Los Alamos National Laboratory, Los Alamos, NM, USA; 3Specifica Inc., Santa Fe, NM, USACorrespondence: Antonietta M LilloUSA Department of Energy, In Care of Los Alamos National Laboratory, Bikini Atoll Road, SM-30, TA-43, Building 0001, Room 220, Los Alamos, NM 87545, USATel +1 505-606-0578Fax +1 505-665-9030Email alillo@lanl.govBackground: Yersinia pestis is a category A infective agent that causes bubonic, septicemic, and pneumonic plague. Notably, the acquisition of antimicrobial or multidrug resistance through natural or purposed means qualifies Y. pestis as a potential biothreat agent. Therefore, high-quality antibodies designed for accurate and sensitive Y. pestis diagnostics, and therapeutics potentiating or replacing traditional antibiotics are of utmost need for national security and public health preparedness.Methods: Here, we describe a set of human monoclonal immunoglobulins (IgG1s) targeting Y. pestis fraction 1 (F1) antigen, previously derived from in vitro evolution of a phage-display library of single-chain antibodies (scFv). We extensively characterized these antibodies and their effect on bacterial and mammalian cells via: ELISA, flow cytometry, mass spectrometry, spectroscopy, and various metabolic assays.Results: Two of our anti-F1 IgG (αF1Ig 2 and αF1Ig 8) stood out for high production yield, specificity, and stability. These two antibodies were additionally attractive in that they displayed picomolar affinity, did not compete when binding Y. pestis, and retained immunoreactivity upon chemical derivatization. Most importantly, these antibodies detected < 1,000 Y. pestis cells in sandwich ELISA, did not harm respiratory epithelial cells, induced Y. pestis agglutination at low concentration (350 nM), and caused apparent reduction in cell growth when radiolabeled at a nonagglutinating concentration (34 nM).Conclusion: These antibodies are amenable to the development of accurate and sensitive diagnostics and immuno/radioimmunotherapeutics.Keywords: immunodiagnostic, radioimmunotherapy, RIT, immunotherapy, radiolabeling, immunoantibiotic, lateral flow assay, LFA

Yersinia pestis, the causative agent of plague and a biological threat agent, presents an urgent need for novel medical countermeasures due to documented cases of naturally acquired antibiotic resistance and potential person-to-person spread during a pneumonic infection. Immunotherapy has been proposed as a way to circumvent current and future antibiotic resistance. Here, we describe the development and characterization of two affinity matured human antibodies (αF1Ig AM2 and αF1Ig AM8) that promote survival of mice after exposure to aerosolized Y. pestis. We share details of the error prone PCR and yeast display technology-based affinity maturation process that we used. The resultant matured antibodies have nanomolar affinity for Y. pestis F1 antigen, are produced in high yield, and are resilient to 37°C stress for up to 6 months. Importantly, in vitro assays using a murine macrophage cell line demonstrated that αF1Ig AM2 and αF1Ig AM8 are opsonic. Even more importantly, in vivo studies using pneumonic plague mouse models showed that 100% of the mice receiving 500 μg of IgGs αF1Ig AM2 and αF1Ig AM8 survived lethal challenge with aerosolized Y. pestis CO92. Combined, these results provide evidence of the quality and robustness of αF1Ig AM2 and αF1Ig AM8 and support their development as potential medical countermeasures against plague., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs 1-3 and revealed how quickly viral escape can curtail effective options 4,5 . When the SARS-CoV-2 Omicron variant emerged in 2021, many antibody drug products lost potency, including Evusheld and its constituent, cilgavimab 4-6 . Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination 4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign and renew the efficacy of COV2-2130 against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and subsequent variants of concern, and provides protection in vivo against the strains tested: WA1/2020, BA.1.1 and BA.5. Deep mutational scanning of tens of thousands of pseudovirus variants reveals that 2130-1-0114-112 improves broad potency without increasing escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Our computational approach does not require experimental iterations or pre-existing binding data, thus enabling rapid response strategies to address escape variants or lessen escape vulnerabilities., (© 2024. The Author(s).)

Here, we describe the isolation of 18 unique anti SARS-CoV-2 human single-chain antibodies from an antibody library derived from healthy donors. The selection used a combination of phage and yeast display technologies and included counter-selection strategies meant to direct the selection of the receptor-binding motif (RBM) of SARS-CoV-2 spike protein's receptor binding domain (RBD2). Selected antibodies were characterized in various formats including IgG, using flow cytometry, ELISA, high throughput SPR, and fluorescence microscopy. We report antibodies' RBD2 recognition specificity, binding affinity, and epitope diversity, as well as ability to block RBD2 binding to the human receptor angiotensin-converting enzyme 2 (ACE2) and to neutralize authentic SARS-CoV-2 virus infection in vitro. We present evidence supporting that: 1) most of our antibodies (16 out of 18) selectively recognize RBD2; 2) the best performing 8 antibodies target eight different epitopes of RBD2; 3) one of the pairs tested in sandwich assays detects RBD2 with sub-picomolar sensitivity; and 4) two antibody pairs inhibit SARS-CoV-2 infection at low nanomolar half neutralization titers. Based on these results, we conclude that our antibodies have high potential for therapeutic and diagnostic applications. Importantly, our results indicate that readily available non immune (naïve) antibody libraries obtained from healthy donors can be used to select high-quality monoclonal antibodies, bypassing the need for blood of infected patients, and offering a widely accessible and low-cost alternative to more sophisticated and expensive antibody selection approaches (e.g. single B cell analysis and natural evolution in humanized mice).

Background: Steroid use in renal transplant is related to multiple adverse effects. Long-term effects of early withdrawal steroids in pediatric renal transplant were assessed., Methods: Renal transplant children with low immunological risk treated on basiliximab, tacrolimus, and mycophenolate with steroid withdrawal or steroid control were evaluated between 2003 and 2019. Clinical variables, treatment adherence, acute rejection, graft loss, and death were analyzed through hazard ratios, and Kaplan-Meier and multivariate analyses., Results: The study included 152 patients, 71.1% steroid withdrawal, mean follow-up 8.5 years, 64.5% structural abnormalities, and 81.6% deceased donor. At 12 years of transplant, event-free survival analysis for graft loss or death showed no significant difference between steroid withdrawal and control steroid treatment (85.9% vs. 80.4%, p = .36) nor in acute rejection at 10 years (18.5% vs. 20.5%, p = .78) or in donor-specific antibody appearance (19.6% vs. 21.4%, p = .98). Delta height Z-score was increased in the steroid withdrawal group (p < .01). The main predictor of graft loss or death was non-adherence to treatment (p = .001; OR: 17.5 [3.3-90.9])., Conclusions: Steroid withdrawal therapy was effective and safe for low-risk pediatric renal transplant in long-term evaluation. Non-adherence was the main predictor of graft loss or death., (© 2021 Wiley Periodicals LLC.)

In vitro display technologies based on phage and yeast have a successful history of selecting single-chain variable fragment (scFv) antibodies against various targets. However, single-chain antibodies are often unstable and poorly expressed in Escherichia coli. Here, we explore the feasibility of converting scFv antibodies to an intrinsically fluorescent format by inserting the monomeric, stable fluorescent protein named thermal green, between the light- and heavy-chain variable regions. Our results show that the scTGP format maintains the affinity and specificity of the antibodies, improves expression levels, allows one-step fluorescent assay for detection of binding and is a suitable reagent for epitope binning. We also report the crystal structure of an scTGP construct that recognizes phosphorylated tyrosine on FcεR1 receptor of the allergy pathway., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Interactions between the cytoplasmic domains of viral transmembrane proteins and host machinery often determine the outcome of viral infection. The M2 protein of influenza A has been identified as a key player in autophagy-mediated viral replication. Here, we describe the engineering and validation of an antibody specific for the cytoplasmic domain of the M2 protein. Through phage and yeast display selection techniques, we obtained an antibody that recognizes: 1) the M2 cytoplasmic domain purified from bacterial inclusion bodies and refolded, 2) full-length M2 recombinant protein expressed in mammalian cells, and 3) native M2 protein in influenza A infected cells. This antibody can serve as a molecular tool to enhance our knowledge of protein-protein interactions between influenza A virus and the host cell machinery. We anticipate the methods described herein will further the development of antibodies specific to the cytoplasmic domains of transmembrane proteins.

Background: Information from historical infectious disease outbreaks provides real-world data about outbreaks and their impacts on affected populations. These data can be used to develop a picture of an unfolding outbreak in its early stages, when incoming information is sparse and isolated, to identify effective control measures and guide their implementation., Objective: This study aimed to develop a publicly accessible Web-based visual analytic called Analytics for the Investigation of Disease Outbreaks (AIDO) that uses historical disease outbreak information for decision support and situational awareness of an unfolding outbreak., Methods: We developed an algorithm to allow the matching of unfolding outbreak data to a representative library of historical outbreaks. This process provides epidemiological clues that facilitate a user's understanding of an unfolding outbreak and facilitates informed decisions about mitigation actions. Disease-specific properties to build a complete picture of the unfolding event were identified through a data-driven approach. A method of analogs approach was used to develop a short-term forecasting feature in the analytic. The 4 major steps involved in developing this tool were (1) collection of historic outbreak data and preparation of the representative library, (2) development of AIDO algorithms, (3) development of user interface and associated visuals, and (4) verification and validation., Results: The tool currently includes representative historical outbreaks for 39 infectious diseases with over 600 diverse outbreaks. We identified 27 different properties categorized into 3 broad domains (population, location, and disease) that were used to evaluate outbreaks across all diseases for their effect on case count and duration of an outbreak. Statistical analyses revealed disease-specific properties from this set that were included in the disease-specific similarity algorithm. Although there were some similarities across diseases, we found that statistically important properties tend to vary, even between similar diseases. This may be because of our emphasis on including diverse representative outbreak presentations in our libraries. AIDO algorithm evaluations (similarity algorithm and short-term forecasting) were conducted using 4 case studies and we have shown details for the Q fever outbreak in Bilbao, Spain (2014), using data from the early stages of the outbreak. Using data from only the initial 2 weeks, AIDO identified historical outbreaks that were very similar in terms of their epidemiological picture (case count, duration, source of exposure, and urban setting). The short-term forecasting algorithm accurately predicted case count and duration for the unfolding outbreak., Conclusions: AIDO is a decision support tool that facilitates increased situational awareness during an unfolding outbreak and enables informed decisions on mitigation strategies. AIDO analytics are available to epidemiologists across the globe with access to internet, at no cost. In this study, we presented a new approach to applying historical outbreak data to provide actionable information during the early stages of an unfolding infectious disease outbreak., (©Nileena Velappan, Ashlynn Rae Daughton, Geoffrey Fairchild, William Earl Rosenberger, Nicholas Generous, Maneesha Elizabeth Chitanvis, Forest Michael Altherr, Lauren A Castro, Reid Priedhorsky, Esteban Luis Abeyta, Leslie A Naranjo, Attelia Dawn Hollander, Grace Vuyisich, Antonietta Maria Lillo, Emily Kathryn Cloyd, Ashvini Rajendra Vaidya, Alina Deshpande. Originally published in JMIR Public Health and Surveillance (http://publichealth.jmir.org), 25.02.2019.)

Protein-ligand conjugations are usually carried out in aqueous media in order to mimic the environment within which the conjugates will be used. In this work, we focus on the conjugation of amphiphilic variants of elastin-like polypeptide (ELP), short elastin (sEL), to poorly water-soluble compounds like OPPVs ( p-phenylenevinylene oligomers), triarylamines, and polypyridine-metal complexes. These conjugations are problematic when carried out in aqueous phase because hydrophobic ligands tend to avoid exposure to water, which in turn causes the ligand to self-aggregate and/or interact noncovalently with hydrophobic regions of the amphiphile. Ultimately, this behavior leads to low conjugation efficiency and contamination with strong noncovalent "conjugates". After exploring the solubility of sEL in various organic solvents, we have established an efficient conjugation methodology for obtaining covalent conjugates virtually free of contaminating noncovalent complexes. When conjugating carboxylated ligands to the amphiphile amines, we demonstrate that even when only one amine (the N-terminus) is present, its derivatization is 98% efficient. When conjugating amine moieties to the amphiphile carboxyls (a problematic configuration), protein multimerization is avoided, 98-100% of the protein is conjugated, and the unreacted ligand is recovered in pure form. Our syntheses occur in "one pot", and our purification procedure is a simple workup utilizing a combination of water and organic solvent extractions. This conjugation methodology might provide a solution to problems arising from solubility mismatch of protein and ligand, and it is likely to be widely applied for modification of recombinant amphiphiles used for drug delivery (PEG-antibodies, polymer-enzymes, food proteins), cell adhesion (collagen, hydrophobins), synthesis of nanostructures (peptides), and engineering of biocompatible optoelectronics (biological polymers), to cite a few.

Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The "helper cell" packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands., Competing Interests: The authors have declared that no competing interests exist.

Background: Fibroblast Growth Factor-23 (FGF23) and cofactor Klotho are key regulators of mineral metabolism in chronic kidney disease (CKD), but little is known about the mechanisms that regulate their production. This study evaluates longitudinal changes of FGF23 and Klotho levels and their regulatory factors in children on chronic peritoneal dialysis (PD)., Methods: FGF23, Klotho, 25(OH) vitamin D, 1,25-dihydroxyvitamin D and parathyroid hormone (PTH) plasma concentrations were measured during 1 year of follow-up in PD children. Anthropometric and dialytical parameters were evaluated in addition to mineral metabolism variables., Results: Thirty-one patients under chronic PD were followed for 12 months. FGF23 mean plasma levels at Month 1 were significantly increased compared with controls, 215.1 ± 303.6 versus 9.4 ± 5.7 pg/mL, respectively (P < 0.001). Baseline Klotho levels were 41% lower in patients compared with controls, 132.1 ± 58 versus 320 ± 119.4 pg/mL, respectively (P < 0.001), and did not correlate with FGF23 and phosphorus levels. At Month 12, FGF23 (195 ± 300 pg/mL) and Klotho levels (130 ± 34 pg/mL) remained similar to baseline values. Log-FGF23 correlated significantly with height/age Z score (r= -0.38) and residual renal function (r = -0.44), but no correlation was found with serum phosphorus, phosphate intake, PTH and vitamin D levels. The log-FGF23 strongly correlated with calcium levels at Months 1, 6 and 12, however, this relationship was blunted if serum phosphorus was >6 mg/dL. By multiple regression analysis, calcium was the strongest variable determining FGF23 levels., Conclusions: In this longitudinal study, FGF23 levels are markedly increased, and Klotho levels are reduced in PD children compared with controls. FGF23 levels appeared to be regulated primarily by serum calcium, showing a significant correlation at each time of measurement. This relationship was lost in patients with phosphorus >6 mg/dL. These observations may have important consequences to the therapeutic management of phosphate homeostasis in CKD patients.

This prospective, comparative trial investigated the impact on mean change in height standard deviation score (SDS), acute rejection rate, and renal function of early steroid withdrawal in 96 recipients with 5 years of follow-up. Recipients under basiliximab induction and steroid withdrawal (SW: TAC/MMF; n = 55) were compared with a matched steroid control group (SC: TAC/MMF/STEROID, n = 41). SW received steroids until Day 6, SC decreased to 10 mg/m(2) within 2 months post-transplant. Five years after SW, the longitudinal growth (SDS) gain was 1.4 ± 0.4 vs. 1.1 ± 0.3 for SC group (p < 0.02). Height benefits in prepubertal and pubertal status in both groups were demonstrated in the delta growth trends (mixed model; p < 0.01). Biopsy-proven acute rejection in SW was 11% and 17.5%, SC (p: ns). Mean eGFR (ml/min/1.73 m(2)) at 5 years post-transplant was SW 80.6 ± 27.8 vs. 82.6 ± 25.1 for SC (p: ns). The death-censored graft survival rate at 1 and 5 years was 99 and 90% for SW; 98 and 96% for SC (p = ns). PTLD incidence in SW 3.3 vs. 2.5% in SC (p: ns). Five years post-transplant, early steroid withdrawal showed positive impacts on growth, stable renal function without increased acute rejection risk, and PTLD incidence.

Objective: To determine the prevalence of voiding dysfunction (VD) in patients with persistent vulvovaginitis (PVV), and to evaluate the clinical response of PVV in the treatment of VD., Patients and Methods: Girls four years or older who consulted for PVV for at least one month and who did not respond to general measures. A physical examination was performed with visual inspection and colposcopy; vaginal samples for culture and vaginoscopy were carried out. On every patient urodynamic studies were performed. Girls who were diagnosed with VD were treated. A pediatric gynecologist did the follow-up; a successful response was considered when inflammatory symptoms and vaginal discharge ceased., Results: Twenty patients were included, mean age 8.6 years (range: 4.6-14 years); 75% prepubertal symptoms lasted for 1.8 years; 19 (95%) had urodynamia, 10 (52.6%) had an overactive bladder, 8 (42.1%) external bladder sphincter dyssynergia, 1 (5.2%) hypotonic bladder, and 13 (65%) showed improvement., Conclusion: VD is an important cause when considering the etiology of PVV., (2011 North American Society for Pediatric and Adolescent Gynecology. Published by Elsevier Inc. All rights reserved.)

Background: Hypovitaminosis D has a high prevalence among patients with chronic kidney disease (CKD)., Aim: To determine the prevalence of 25 hydroxy vitamin D (25(OH) D) insufficiency and deficiency in pediatric patients on dialysis and kidney transplantation., Material and Methods: Serum calcium and phosphorus, parathormone (PTH), alkaline phosphatases and 25 (OH)D were measured in 13 children on hemodialysis (HD), 18 on peritoneal dialysis (PD) and 53 that received an allograft (Tx), aged 9.8 ± 4.6 years (51% females)., Results: Fifty four percent of patients had height Z score less than -1.88. Patients on HD had the lowest values. The average time of replacement therapy was 2.9 ± 2.8 years. Mean 25(OH)D levels in all was 18.7 ± 10.7ng/ml (HD: 21 ± 16.8, PD: 18.9 ± 8.5, Tx: 18.1 ± 9.72 ng/ml). Eighty eight percent of patients had levels below 30 ng/ml. Mean of serum calcium was 9.5 ± 0.64 mg/dl, serum phosphorus 5.03 ± 1.02 mg/dl, calcium-phosphorus product 48 ± 11.8 mg/dl and alkaline phosphatases 300.5 ± 171.3 IU/L. Average PTH values in dialyzed and Tx patients were 724.6 ± 640.5 and 107.7 ± 56.2 pg/ml, respectively (p < 0.001). A positive correlation between 25 (OH) D and calcium levels among PD patients was observed (r = 0.490, p = 0.04)., Conclusions: Hypovitaminosis D is highly prevalent among children on renal substitution therapy, regardless of the type of therapy used and the stage of renal failure.

Background: Most Yersinia pestis strains are known to express a capsule-like antigen, fraction 1 (F1)(.) F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is found in Y. pestis but not in closely related species such as Yersinia enterocolytica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody fragment (scFv) phage display library using purified F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection., Methods: Phage antibody panning was performed against F1 in an automated fashion using the Kingfisher magnetic bead system. Selected scFvs were screened for F1-binding specificity by one-step alkaline phosphatase enzyme linked immunosorbant assay (ELISA), and assayed for binding to recombinant antigen and/or Y. pestis by flow cytometry and whole-cell ELISA., Results: Seven of the eight selected scFvs were shown to specifically bind both recombinant F1 and a panel of F1-positive Yersinia cells. The majority of the soluble scFvs were found to be difficult to purify, unstable and prone to cross-reactivity with F1-negative Yersinia strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA., Conclusions: Our high throughput methods of selection and screening allowed for time and cost effective discovery of seven scFvs specifically binding Y. pestis F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv antibodies.

Steroids have been a cornerstone in renal transplant immunosuppression despite cardiovascular risk and growth impairment in children. New immunosuppressive drugs have allowed early withdrawal or even complete avoidance of steroids. To evaluate a new immunosuppressive protocol with early withdrawal of steroids in a pediatric renal transplant population, we initiated a prospective study in recipients >1 year old who showed low immunologic risk was started. Group A (n = 12) received decreasing doses of steroids until day posttransplant 7 under a regimen of Tacrolimus (FK) and mycophenolate mofetil (MMF). Group B (n = 11) were controls treated with steroids, cyclosporine and azathioprine. In both groups, induction therapy included basiliximab. We evaluated anthropometric and biochemical variables, acute rejection episodes (ARE), and cytomegalovirus (CMV) infection. Mean values and variations for continuous variables were calculated at months 1 and 3 for comparison at the same time using student's t-test and regresion analysis. We obtained mean values at months 1, 3, and 6 for groups A and B of creatinine clearance (mL/min): 86.2 versus 107.4; 76.9 versus 96.6; 73.3 versus 97.9 (P < .05); hematocrit (%) was 27.4 versus 31.8; 29.3 versus 33.9; 32.9 versus 34.3% (P < .05); total cholesterol (mg/dL), 148 versus 195, 139 versus 85, 142 versus 174 (P < .05); creatinine clearance decreased in both groups during follow-up with a smaller slope among group A (P < .05). No differences were observed between the groups in Z height, diastolic and systolic blood pressures at 6 months of follow-up. Serum total cholesterol mean levels at months 1, 3, and 6 were significantly lower among the group withdrawn from steroids (P < .05). Plasma bicarbonate levels were lower among group A than B; there was no difference in blood glucose levels. No AREs and no difference in CMV infections were observed. In conclusion, early withdrawal of steroids with FK and MMF was not associated with a higher incidence of either ARE or CMV infection. Lower levels of cholesterol could imply a reduced cardiovascular risk. Longer follow-up is needed to evaluate the impact of this therapy on renal function and linear growth.

Steroids have been a cornerstone in renal transplant immunosuppression. New immunosuppressive drugs have led to protocols using early steroid withdrawal or complete avoidance. A prospective protocol in 23 pediatric renal transplant (ages 2-14 yr) who received decreasing steroid doses stopping at day 7 post-Tx, FK, and MMF were compared with a CsA, AZT, historically matched steroid-based control group. Basiliximab was used in two doses. Anthropometric, biochemical variables, AR rates, and CMV infection were evaluated and compared using Student's t-test and regression analysis. A better growth pattern was seen in steroid withdrawal group. GFR rate and serum glucose were similar in both groups. Total serum cholesterol levels were significantly lower in steroid withdrawal group. The incidence of AR at 12 months was 4.3% in steroid withdrawal group vs. 8.6% in steroid-based group (p = ns). No difference in CMV infection was observed. Hemoglobin levels were low during the first months in both groups; reached normal values after six months. SBP became higher at 12 months in steroid-based group. Patient and graft survival was 98% in both groups at one-yr post-transplant. Early steroid withdrawal was efficacious, safe, and did not increase risk of rejection, preserving optimal growth, renal function, and reducing cardiovascular risk factors.

Background: Calcitriol has long been used as the main therapy in renal osteodystrophy, but the efficacy of the oral route is not always as high as expected., Objective: To asses the safety and efficacy of intraperitoneal calcitriol in infants undergoing peritoneal dialysis (PD)., Patients and Methods: PD patients on oral calcitriol therapy, with serum parathyroid hormone (PTH) >1000 pg/mL during the previous 3 months of treatment, were switched to intraperitoneal calcitriol therapy, 1 microg twice per week. Dose was increased to 1 microg three times per week if PTH remained >1000 pg/mL, and was later readjusted. Target PTH was 200-300 pg/mL according DOQI guidelines., Statistics: All results are expressed as mean +/- SE. The Wilcoxon signed rank test was used to evaluate differences in measurements for each pair of values. The confidence interval for differences between population medians was 96.9%. A p value less than 0.05 was considered significant., Results: Six male children, mean age 17 +/- 3.86 months, completed a 12-month follow-up. Mean pretreatment PTH was 1654 +/- 209 pg/mL. Mean PTH at months 0, 3, 6, 9, and 12 was 1448 +/- 439*, 1277 +/- 723, 910 +/- 704, 582 +/- 282*, and 465 +/- 224* pg/mL, respectively (*p < 0.05). Twelve hypercalcemic and 10 hyperphosphatemic episodes were successfully treated., Conclusion: Infants on PD who fail to respond to oral calcitriol therapy can be safely treated with intraperitoneal administration of active vitamin D.

Peptidic ligands can be used for specific cell targeting and the delivery of payloads into the target cell. Here we describe the screening of a pool of cyclic peptide phage display libraries using whole-cell panning against human melanoma cell line Me6652/4. This strategy resulted in the selection of the cyclic 13-mer Pep42, CTVALPGGYVRVC, which showed preferential internalization into melanoma cell line Me6652/4 versus the reference cell line Me6652/56. This translocation is a receptor-mediated process that does not require electrostatic interactions nor does it involve transfer to the lysosomal compartment. The cellular receptor for Pep42 was identified as the surface membrane form of glucose-regulated protein 78 (GRP78), a member of the heat shock protein family and a marker on malignant cancer cells. The cellular uptake and intracellular trafficking of Pep42-Quantum Dot conjugates was monitored by confocal laser microscopy, and colocalization within the endoplasmic reticulum was observed. The uptake of Pep42 could be blocked by a monoclonal antibody against the identified receptor. Furthermore, Pep42 was shown to target specifically GRP78-expressing cancer cells. The in vitro cytotoxicity of a Pep42-Taxol conjugate was evaluated by flow cytometry wherein the conjugate was shown to induce apoptosis and was more effective in promoting programmed cell death in Me6652/4 cells. In summary, the data presented suggest that cyclic peptide Pep42 might be a powerful tool in the construction of drug conjugates designed to selectively kill malignant cancer cells.

Between 1989 and 2003, 100 transplants were performed in 96 patients at the pediatric nephrology unit of the Calvo Mackenna Children's Hospital. Mean age 10.9 +/- 3.9 yr (1-17.6), 30% from LD. Donors were younger than 5 yr in five patients and all recipients received an 'en bloc' graft. Original disease was hypo/dysplasia 27%, reflux nephropathy 22 and 17% chronic glomerulonephritis. The immunosuppressive protocol during the first period (n = 56, 1989-2000): Cyclosporine, steroids and azathioprine, and during the second period (n = 44, 2001-2003): FK, steroids, MMF and anti-CD25 antibody (mAbs). AR was reported in 22 patients, 11% in LD, 31% in DD (p < 0.01). The AR rate decreased from 40 to 8% after anti-CD25 monoclonal induction. Patient actuarial survival rate at 1, 3 and 5 yr was 100% for LD and 96% for DD. The overall actuarial graft survival at 1,3, and 5 yr was 96.7, 96.7 and 71% for LD and 89, 76 and 73% for DD donors. Graft survival rate improved from the first period (1989-2000) to the second period (2001-2003; p = 0.05). No difference in graft survival rate with HLA-A,B,DR matching was found. Graft survival rate was better when cold ischemia time was <24 h (p < 0.01). CMV infections increased from 19 to 40% when MMF and anti-CD25 Ab were introduced (p < 0.01). The height/age Z score at 1, 3 and 5 yr post-transplant was -2.2, -2.1, -2.2, respectively, for children older than 7 yr and -1.8, -1.9, -2.1 for those transplanted younger than 7 yr of age who were switched to alternate day steroids (p < 0.01). The cause of graft lost was: chronic rejection eight, non-adherence four, AR four and vascular thrombosis two. The cause of death in two patients was fungus septicemia and accelerated rejection. Pediatric renal transplantation can be performed in our group with acceptable morbidity, low mortality and graft survival rates similar to other reports in North America and Western Europe. Graft survival rate improved with newer immunosuppression and greater experience at the center. Management of non-adherence and chronic rejection remain the major challenges.

Escherichia coli 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (YgbP or IspD) catalyzes the conversion of 2-C-methyl-D-erythritol 4-phosphate (MEP) and cytidine triphosphate (CTP) to 4-diphosphocytidyl-2-C-methylerythritol (CDPME). Pulse chase experiments established that the reaction involves an ordered sequential mechanism with mandatory initial binding of CTP. On the basis of analysis of the previously reported crystal structures of apo-YgbP as well as YgbP complexed with both CTP.Mg(2+) and CDPME.Mg(2+) [Richard, S. B., Bowman, M. E., Kwiatkowski, W., Kang, I., Chow, C., Lillo, A. M., Cane, D. E., and Noel, J. P. (2001) Nat. Struct. Biol. 8, 641-648], a group of active site residues were selected for site-directed mutagenesis and steady-state kinetic analysis. Both Lys27 and Lys213 were shown to be essential to catalytic activity, consistent with their proposed role in stabilization of a pentacoordinate phosphate transition state resulting from in-line attack of the MEP phosphate on the alpha-phosphate of CTP. In addition, Thr140, Arg109, Asp106, and Thr165 were all shown to play critical roles in the binding and proper orientation of the MEP substrate.

Selective antitumor chemotherapy can be achieved by using antibody-drug conjugates that recognize surface proteins upregulated in cancer cells. One such receptor is integrin alpha3beta1, which is overexpressed on malignant melanoma, prostate carcinoma, and glioma cells. We previously identified a human single-chain Fv antibody (scFv), denoted Pan10, specific for integrin alpha3beta1 that is internalized by human pancreatic cancer cells. Herein, we describe the chemical introduction of reactive thiol groups onto Pan10, the specific conjugation of the modified scFv to maleimide-derivatized analogs of the potent cytotoxic agent duocarmycin SA, and the properties of the resultant conjugates. Our findings provide evidence that Pan10-drug conjugates maintain the internalizing capacity of the parent scFv and are cytotoxic at nanomolar concentrations. Our Pan10-drug conjugates may be promising candidates for targeted chemotherapy of malignant diseases associated with overexpression of integrin alpha3beta1.

The eryA gene of the bacterial pathogen Brucella abortus has been functionally expressed in Escherichia coli. The resultant EryA was shown to catalyze the ATP-dependent conversion of erythritol to L-erythritol-4-phosphate (L-E4P). The steady state kinetic parameters of this reaction were determined and the enzyme was used to prepare L-E4P which was shown to be a weak inhibitor of 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (YgbP).

The enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) synthase catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to MECDP, a highly unusual cyclodiphosphate-containing intermediate on the mevalonate-independent pathway to isopentenyl diphosphate and dimethylallyl diphosphate. We now report two x-ray crystal structures of MECDP synthase refined to 2.8-A resolution. The first structure contains a bound Mn(2+) cation, and the second structure contains CMP, MECDP, and Mn(2+). The protein adopts a homotrimeric quaternary structure built around a central hydrophobic cavity and three externally facing active sites. Each of these active sites is located between two adjacent monomers. A tetrahedrally arranged transition metal binding site, potentially occupied by Mn(2+), sits at the base of the active site cleft. A phosphate oxygen of MECDP and the side chains of Asp(8), His(10), and His(42) occupy the metal ion coordination sphere. These structures reveal for the first time the structural determinants underlying substrate, product, and Mn(2+) recognition and the likely catalytic mechanism accompanying the biosynthesis of the cyclodiphosphate-containing isoprenoid precursor, MECDP.

The YgbP protein of Escherichia coli encodes the enzyme 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) synthetase, a member of the cytidyltransferase family of enzymes. CDP-ME is an intermediate in the mevalonate-independent pathway for isoprenoid biosynthesis in a number of prokaryotic organisms, algae, the plant plastids and the malaria parasite. Because vertebrates synthesize isoprenoid precursors using a mevalonate pathway, CDP-ME synthetase and other enzymes of the mevalonate-independent pathway for isoprenoid production represent attractive targets for the structure-based design of selective antibacterial, herbicidal and antimalarial drugs. The high-resolution structures of E. coli CDP-ME synthetase in the apo form and complexed with both CTP-Mg2+ and CDP-ME-Mg2+ reveal the stereochemical principles underlying both substrate and product recognition as well as catalysis in CDP-ME synthetase. Moreover, these complexes represent the first experimental structures for any cytidyltransferase with both substrates and products bound.