Multimaterial actinic spatial control 3D and 4D printing.

Production of objects with varied mechanical properties is challenging for current manufacturing methods. Additive manufacturing could make these multimaterial objects possible, but methods able to achieve multimaterial control along all three axes of printing are limited. Here we report a multi-wavelength method of vat photopolymerization that provides chemoselective wavelength-control over material composition utilizing multimaterial actinic spatial control (MASC) during additive manufacturing. The multicomponent photoresins include acrylate- and epoxide-based monomers with corresponding radical and cationic initiators. Under long wavelength (visible) irradiation, preferential curing of acrylate components is observed. Under short wavelength (UV) irradiation, a combination of acrylate and epoxide components are incorporated. This enables production of multimaterial parts containing stiff epoxide networks contrasted against soft hydrogels and organogels. Variation in MASC formulation drastically changes the mechanical properties of printed samples. Samples printed using different MASC formulations have spatially-controlled chemical heterogeneity, mechanical anisotropy, and spatially-controlled swelling that facilitates 4D printing.

Prolonged-Release vs Immediate-Release Tacrolimus Capsules in Black vs White Kidney Transplant Patients: A Post Hoc Analysis of Phase III Data.

BACKGROUND: Black kidney transplant patients experience inferior outcomes compared with other ethnicities. Because scrutiny is required when immunosuppressant drugs are used in such at-risk populations, we report the first large-scale clinical efficacy data assessing prolonged-release tacrolimus (PR-T) in black de novo kidney transplant patients. METHODS AND MATERIALS: We used logistic regression and proportionate hazards to compare a composite outcome measure (biopsy-proven acute rejection, graft loss, mortality, and loss to follow-up) in black and white patients in treatment groups longer than 24 weeks, from 3 large Phase III randomized controlled trials. Secondary endpoints included tacrolimus trough concentration, dose, and estimated glomerular filtration rate. RESULTS: The study included 2162 patients whose treatments belonged to two categories (immediate-release tacrolimus: 77 black patients, 721 white patients; and PR-T: 87 black patients, 1277 white patients). Despite demographic factors generally predictive of worse outcomes, efficacy failure among black patients who received PR-T was non-inferior to that among white patients who received either therapy. Compared with immediate-release tacrolimus, black patients who received PR-T achieved stable tacrolimus concentrations 2.5 times faster (21 vs 56 days, P = .04), and more achieved stable target concentrations (76.7% vs 69.3%). Treatment-emergent adverse events were consistent with those reported separately in pivotal trials. CONCLUSIONS: Overall, black patients who received PR-T achieved non-inferior outcomes compared to white patients, despite higher pretransplant risk among black patients. Moreover, PR-T improved the time to achieve, and the likelihood of reaching, stable therapeutic concentrations among black patients, suggesting that PR-T could improve the consistency of tacrolimus exposure in this patient population.

Micropollutants in urban stormwater: occurrence, concentrations, and atmospheric contributions for a wide range of contaminants in three French catchments.

This study aimed at: (a) providing information on the occurrence and concentration ranges in urban stormwater for a wide array of pollutants (n = 77); (b) assessing whether despite the differences between various catchments (land use, climatic conditions, etc.), the trends in terms of contamination level are similar; and (c) analyzing the contribution of total atmospheric fallout (TAF) with respect to sources endogenous to this contamination. The studied contaminants include conventional stormwater contaminants (polycyclic aromatic hydrocarbons (PAHs), Zn, Cu, Pb, etc.), in addition to poorly or undocumented pollutants such as nonylphenol and octylphenol ethoxylates (NPnEO and OPnEO), bisphenol A (BPA), polybrominated diphenyl ethers (PBDEs), a wide variety of pesticides, and various metals of relevance (As, Ti, Sr, V). Sampling and analysis were performed using homogeneous methods on three urban catchments with different land use patterns located in three distinct French towns. For many of these pollutants, the results do not allow highlighting a significant difference in stormwater quality at the scale of the three urban catchments considered. Significant differences were, however, observed for several metals (As, Cr, Cu, Ni, Sr and Zn), PAHs, and PBDEs, though this assessment would need to be confirmed by further experiments. The pollutant distributions between dissolved and particulate phases were found to be similar across the three experimental sites, thus suggesting no site dependence. Lastly, the contributions of TAF to stormwater contamination for micropollutants were quite low. This finding held true not only for PAHs, as previously demonstrated in the literature, but also for a broader range of molecules such as BPA, NPnEO, OPnEO, and PBDEs, whose high local production is correlated with the leaching of urban surfaces, buildings, and vehicles.

Use of argatroban during multi-organ procurement: pharmacokinetics and sequelae in recipient of transplanted liver.

This report illustrates potential concerns regarding the administration of Argatroban (AGN), a small molecule, direct thrombin inhibitor, within the setting of multi-organ procurement (MOP). Herein, we outline the case of a large AGN bolus to the donor during MOP, and the passive transfer of a coagulopathy to the recipient of the transplanted liver. From this, we conclude that caution should be exercised when AGN is used in the setting of MOP.

The effect of precursor structures on the action of glucosaminyl 3-O-sulfotransferase-1 and the biosynthesis of anticoagulant heparan sulfate.

To understand how 2-O-sulfation of uronic acid residues influences the biosynthesis of anticoagulant heparan sulfate, the cDNA encoding glucosaminyl 3-O-sulfotransferase-1 (3-OST-1) was introduced into wild-type Chinese hamster ovary cells and mutant pgsF-17 cells, which are defective in 2-O-sulfation. 3-OST-1-transduced cells gained the ability to bind to antithrombin. Structural analysis of the heparan sulfate chains showed that 3-OST-1 generates sequences containing GlcUA-GlcN(SO(3))3(SO(3)) and GlcUA-GlcN(SO(3))3(SO(3))6(SO(3)) in both wild-type and mutant cells. In addition, IdoUA-GlcN(SO(3))3(SO(3)) and IdoUA-GlcN(SO(3))3(SO(3))6(SO(3)) accumulate in the mutant chain. These disaccharides were also observed by tagging [6-(3)H]GlcN-labeled pgsF-17 heparan sulfate in vitro with [(35)S]PAPs and purified 3-OST-1. Heparan sulfate derived from the transduced mutant also had approximately 2-fold higher affinity for antithrombin than heparan sulfate derived from the transduced wild-type cells, and it inactivated factor Xa more efficiently. This study demonstrates for the first time that (i) 3-O-sulfation by 3-OST-1 can occur independently of the 2-O-sulfation of uronic acids, (ii) 2-O-sulfation usually occurs before 3-O-sulfation, (iii) 2-O-sulfation blocks the action of 3-OST-1 at glucosamine residues located to the reducing side of IdoUA units, and (iv) that alternative antithrombin-binding structures can be made in the absence of 2-O-sulfation.

Gas 6 promotes Axl-mediated survival in pulmonary endothelial cells.

We examined Gas 6-Axl interactions in human pulmonary artery endothelial cells (HPAEC) and in Axl-transduced HPAEC to test Gas 6 function during endothelial cell survival. We identified the 5.0-kb Axl, 4.2-kb Rse, and 2.6-kb Gas 6 mRNAs in HPAEC. Immunoprecipitation and Western blotting confirmed the presence of these proteins. Gas 6 is present in cell-associated and secreted fractions of growth-arrested HPAEC, independent of cell density. In addition, the Axl receptor is constitutively phosphorylated in growth-arrested cultures, and exogenous Gas 6 enhanced Axl phosphorylation threefold. Gas 6 added to growth-arrested HPAEC resulted in a significant increase in cell number (1.5 nM Gas 6 increased cell number 35%). Flow cytometry revealed that Gas 6 treatment resulted in 28% fewer apoptosing cells. Transduction of a full-length Axl cDNA into HPAEC resulted in 54% fewer apoptosing cells after Gas 6 treatment. Collectively, the data demonstrate antiapoptotic activities for Gas 6 in HPAEC and suggest that Gas 6 signaling may be relevant to endothelial cell survival in the quiescent environment of the vessel wall.

Peptide-mediated cellular delivery.

Peptide-mediated molecular therapeutic delivery systems have recently emerged as an alternative means to effectively substitute or augment present gene therapy technologies, e.g., TAT, VP22, engineered peptides. These systems show great promise for the elimination of the main bottleneck to safe, efficient, targeted gene therapy delivery and are able to efficiently introduce DNA, antisense peptide nucleic acids, oligonucleotides, small molecules and proteins into cells both in vitro and in vivo. They are versatile and easily designed to incorporate a number of specific attributes required for efficient cargo delivery. A fundamentally new property of these moieties will allow the therapeutic intervention in the biochemistry of the target cell without the need to alter its genome.

The role of syndecan cytoplasmic domain in basic fibroblast growth factor-dependent signal transduction.

To determine the role played by syndecan-4 cytoplasmic domain in the mediation of basic fibroblast growth factor (bFGF) signaling, immortalized human cells (ECV) were used to generate cell lines expressing constructs encoding full-length sequences for syndecan-4 (S4), syndecan-1 (S1), glypican-1 (G1), or chimeric proteins consisting of the ectoplasmic domain of glypican-1 linked to the transmembrane/cytoplasmic domain of syndecan-4 (G1-S4c) and the ectoplasmic domain of syndecan-4 linked to the glypican-1 glycosylphosphatidylinositol (GPI) anchor sequence (S4-GPI). Vector-transduced cells (VC) were used as controls. Expression of all these proteoglycans (except for the vector control) significantly increased cell-associated heparan sulfate mass and the number of low affinity bFGF-binding sites. However, in low serum medium, the addition of bFGF stimulated growth and migration of cells expressing S4 and G1-S4c constructs but not G1, S1, S4-GPI, or VC cells. Similar results were obtained using Matrigel growth assays. Mutations of heparan sulfate attachment sites on S4 construct abolished syndecan-4-dependent augmentation of bFGF responses. We conclude that cytoplasmic tail of syndecan-4 plays an important role in bFGF-mediated signal transduction.

Expression of heparan sulfate D-glucosaminyl 3-O-sulfotransferase isoforms reveals novel substrate specificities.

The 3-O-sulfation of glucosamine residues is an important modification during the biosynthesis of heparan sulfate (HS). Our previous studies have led us to purify and molecularly clone the heparan sulfate D-glucosaminyl 3-O-sulfotransferase (3-OST-1), which is the key enzyme converting nonanticoagulant heparan sulfate (HSinact) to anticoagulant heparan sulfate (HSact). In this study, we expressed and characterized the full-length cDNAs of 3-OST-1 homologous genes, designated as 3-OST-2, 3-OST-3A, and 3-OST-3B as described in the accompanying paper (Shworak, N. W., Liu, J., Petros, L. M., Zhang, L., Kobayashi, M., Copeland, N. G., Jenkins, N. A., and Rosenberg, R. D. (1999) J. Biol. Chem. 274, 5170-5184). All these cDNAs were successfully expressed in COS-7 cells, and heparan sulfate sulfotransferase activities were found in the cell extracts. We demonstrated that 3-OST-2, 3-OST-3A, and 3-OST-3B are heparan sulfate D-glucosaminyl 3-O-sulfotransferases because the enzymes transfer sulfate from adenosine 3'-phosphophate 5'-phospho-[35S]sulfate ([35S]PAPS) to the 3-OH position of glucosamine. 3-OST-3A and 3-OST-3B sulfate an identical disaccharide. HSact conversion activity in the cell extract transfected by 3-OST-1 was shown to be 300-fold greater than that in the cell extracts transfected by 3-OST-2 and 3-OST-3A, suggesting that 3-OST-2 and 3-OST-3A do not make HSact. The results of the disaccharide analysis of the nitrous acid-degraded [35S]HS suggested that 3-OST-2 transfers sulfate to GlcA2S-GlcNS and IdoA2S-GlcNS; 3-OST-3A transfers sulfate to IdoA2S-GlcNS. Our results demonstrate that the 3-O-sulfation of glucosamine is generated by different isoforms depending on the saccharide structures around the modified glucosamine residue. This discovery has provided evidence for a new cellular mechanism for generating a defined saccharide sequence in structurally complex HS polysaccharide.

Expression cloning of LDLB, a gene essential for normal Golgi function and assembly of the ldlCp complex.

The Chinese hamster ovary (CHO) cell mutants ldlC and ldlB, which exhibit almost identical phenotypes, define two genes required for multiple steps in the normal medial and trans Golgi-associated processing of glycoconjugates. The LDLC gene encodes ldlCp, an approximately 80-kDa protein, which in wild-type, but not ldlB, cells associates reversibly with the cytoplasmic surface of the Golgi apparatus. Here, we have used a retrovirus-based expression cloning system to clone a murine cDNA, LDLB, that corrects the pleiotropic mutant phenotypes of ldlB cells. The corresponding mRNA was not detected in ldlB mutants. LDLB encodes an approximately 110-kDa protein, ldlBp, which lacks homology to known proteins and contains no common structural motifs. Database searches identified short segments of homology to sequences from Drosophila melanogaster, Arabidopsis thaliana, and Caenorhabditis elegans, and the essentially full-length homologous human sequence (82% identity); however, as was the case for ldlCp, no homologue was identified in Saccharomyces cerevisiae. We have found that in wild-type cell cytosols, ldlCp is a component of an approximately 950-kDa "ldlCp complex," which is smaller, approximately 700 kDa, in ldlB cytosols. Normal assembly of this complex is ldlBp-dependent and may be required for Golgi association of ldlCp and for the normal activities of multiple luminal Golgi processes.

The retinoic acid and cAMP-dependent up-regulation of 3-O-sulfotransferase-1 leads to a dramatic augmentation of anticoagulantly active heparan sulfate biosynthesis in F9 embryonal carcinoma cells.

Retinoic acid (RA) and dibutyryl cAMP plus theophilline (CT) trigger F9 cells to differentiate into parietal endoderm. The differentiation induces a 9-fold increase in total heparan sulfate (HStotal) biosynthesis and a 170-fold increase in anticoagulantly active HS (HSact) biosynthesis. Measurement of 3-O-sulfotransferase-1 mRNA and enzymatic activity demonstrated an increase of over 100-fold whereas determination of N-, 2-O, and 6-O-sulfotransferase enzymatic activities showed elevations of 2-, 3. 5-, and 3.7-fold, respectively. HSact precursor pool measurements reveal that 30% of control F9 HStotal can be converted into HSact while only an additional 10% of RACT F9 HStotal can be transformed into HSact. Disaccharide analysis of metabolic labeled HS indicated that 32% 3-O-sulfate containing disaccharides, i.e. GlcA-anManR3S and GlcA-anManR3S6S, are present in HSact and 68% GlcA-anManR3S and GlcA-anManR3S6S are found in anticoagulantly inactive HS (HSinact). By using adenosine 3'-phosphate 5'-phosphosulfate and purified 3-O-sulfotransferase-1, 30% of 3-O-sulfation occurs in HSact and 70% of 3-O-sulfation occurs in HSinact. The similar ratio of 3-O-sulfate distribution in HSact versus HSinact suggests that HSact production in the F9 system is determined by the abundance of 3-O-sulfotransferase-1 as well as the size of the HSact precursor pool. Extensively 3-O-sulfated HSinact may play an important functional role under in vivo conditions within the murine placenta.

Molecular cloning and expression of mouse and human cDNAs encoding heparan sulfate D-glucosaminyl 3-O-sulfotransferase.

The cellular rate of anticoagulant heparan sulfate proteoglycan (HSPGact) generation is determined by the level of a kinetically limiting microsomal activity, HSact conversion activity, which is predominantly composed of the long sought heparan sulfate D-glucosaminyl 3-O-sulfotransferase (3-OST) (Shworak, N. W., Fritze, L. M. S., Liu, J., Butler, L. D., and Rosenberg, R. D. (1996) J. Biol. Chem. 271, 27063-27071; Liu, J., Shworak, N. W., Fritze, L. M. S., Edelberg, J. M., and Rosenberg, R. D. (1996) J. Biol. Chem. 271, 27072-27082). Mouse 3-OST cDNAs were isolated by proteolyzing the purified enzyme with Lys-C, sequencing the resultant peptides as well as the existing amino terminus, employing degenerate polymerase chain reaction primers corresponding to the sequences of the peptides as well as the amino terminus to amplify a fragment from LTA cDNA, and utilizing the resultant probe to obtain full-length enzyme cDNAs from a lambda Zap Express LTA cDNA library. Human 3-OST cDNAs were isolated by searching the expressed sequence tag data bank with the mouse sequence, identifying a partial-length human cDNA and utilizing the clone as a probe to isolate a full-length enzyme cDNA from a lambda TriplEx human brain cDNA library. The expression of wild-type mouse 3-OST as well as protein A-tagged mouse enzyme by transient transfection of COS-7 cells and the expression of both wild-type mouse and human 3-OST by in vitro transcription/translation demonstrate that the two cDNAs directly encode both HSact conversion and 3-OST activities. The mouse 3-OST cDNAs exhibit three different size classes because of a 5'-untranslated region of variable length, which results from the insertion of 0-1629 base pairs (bp) between residues 216 and 217; however, all cDNAs contain the same open reading frame of 933 bp. The length of the 3'-untranslated region ranges from 301 to 430 bp. The nucleic acid sequence of mouse and human 3-OST cDNAs are approximately 85% similar, encoding novel 311- and 307-amino acid proteins of 35,876 and 35,750 daltons, respectively, that are 93% similar. The encoded enzymes are predicted to be intraluminal Golgi residents, presumably interacting via their C-terminal regions with an integral membrane protein. Both 3-OST species exhibit five potential N-glycosylation sites, which account for the apparent discrepancy between the molecular masses of the encoded enzyme (approximately 34 kDa) and the previously purified enzyme (approximately 46 kDa). The two 3-OST species also exhibit approximately 50% similarity with all previously identified forms of the heparan biosynthetic enzyme N-deacetylase/N-sulfotransferase, which suggests that heparan biosynthetic enzymes share a common sulfotransferase domain.

Analysis of 2-difluoromethyl-DL-ornithine in human plasma, cerebrospinal fluid and urine by cation-exchange high-performance liquid chromatography.

An analytical method has been developed based on cation-exchange liquid chromatography for the measurement of 2-difluoromethyl-DT-ornithine (DFMO) in human plasma, cerebrospinal fluid (CSF) and urine. Fluorescence detection at excitation/emission wavelengths of 340/440 nm is followed by postcolumn derivatization with o-phthalaldehyde-2,mercaptoethanol. All calibration ranges yielded linear relationships with correlation coefficients better than 0.999. In each case the limit of quantitation was equal to the lowest value of the standard curve. The variability of the assay, expressed as relative standard deviations, was less than 7.1%, 15.3% and 7.1% for plasma, CSF and urine, respectively. The accuracy of the assay (expressed as relative errors) ranged between 4.3% and 2.0% for plasma analysis, between -0.1% and 14.0% for CSF analysis and between -8.0% and 2.0% for urine analysis. Plasma, CSF and urinary DFMO concentrations were measured in samples obtained from patients undergoing treatment for trypanosomiasis. The method was found to be applicable for the measurement of DFMO levels in human body fluids for the determination of pharmacokinetic parameters in clinical studies.

Simultaneous measurement of dolasetron and its major metabolite, MDL 74,156, in human plasma and urine.

A selective and sensitive analytical method for the simultaneous measurement of dolasetron (I) and its major metabolite, MDL 74,156 (II), in human plasma and urine samples has been developed using a structural analogue. MDL 101,858, as internal standard (I.S.). The compounds were extracted from plasma and urine using solvent extraction after the addition of the I.S. Chromatographic separation was carried out on a reversed-phase HPLC column and detection and quantification was by fluorescence with excitation and emission wavelengths of 285 and 345 nm, respectively. Linear responses were obtained over concentration ranges of 5 to 1000 pmol/ml for plasma samples and 20 to 1000 pmol/ml for urine samples with correlation coefficients for the calibration curves exceeding 0.999 in all cases. Intra-day and inter-day reproducibility yielded limits of quantification of 10 pmol/ml for I and 5 pmol/ml for II plasma and 50 pmol/ml for I and II in urine. The method has been applied to the simultaneous analysis of both compounds in plasma and urine samples coming from clinical pharmacokinetic studies.

Sequence analysis of the ribosomal RNA operon of the Lyme disease spirochete, Borrelia burgdorferi.

An 11,955-bp region of the Borrelia burgdorferi chromosome containing all the genes encoding ribosomal RNA (rRNA) has been sequenced. The region contains a single gene encoding 16S rRNA and two genes encoding the 23S and 5S rRNAs. The sizes of the 16S, 23S and 5S rRNAs encoded by these genes are 1537, 2926 and 112 nucleotides, respectively. In addition, the genes encoding tRNA(Ala) and tRNA(Ile) are located in the intergenic spacer between the 16S and 23S rDNAs. The tDNAs do not encode the common CCA 3' end which presumably must be added posttranscriptionally. All the genes are present in the same orientation, except for that encoding tRNA(Ile), which is transcribed from the opposite strand. The latter implies that the rDNAs are not transcribed as a single unit. The location of putative promoters and termination signals in the sequence suggest that the 16S rRNA and tRNA(Ala) are transcribed as a single unit, tRNA(Ile) is produced as an individual transcript and the 23S and 5S rDNAs are co-transcribed. Several of the features of this rDNA organization are unique, not having been described previously in any other eubacteria.

Base sequence selectivity in the binding of 7(R),8(S)-dihydroxy-9(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to oligodeoxyribonucleotide duplexes.

The effect of nucleotide sequence on the binding of 7(R),8(S)-dihydroxy-9(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE] to the exocyclic amino group of deoxyguanosine was investigated in duplexes formed by self-complementary oligodeoxyribonucleotide decamers which contained two deoxyguanosines (dGs) within unique sequences. A 35S-postlabeling procedure was developed for analysis of (+)-anti-BPDE adducts as dinucleotides containing 5'-(+)-anti-BPDE-dG adducts. This allows identification of the 3' neighbor of the reacted guanine and permits quantitation of the binding of (+)-anti-BPDE to each specific guanine in the oligodeoxyribonucleotide duplexes. Of all the central dG-containing sequences studied, dG surrounded by deoxycytidines (CGC) reacted to the greatest extent: over 4-fold more (+)-anti-BPDE bound to this central dG compared to the least reactive deoxyguanosine (AGT). (+)-anti-BPDE exhibited a preference for binding to a central deoxyguanosine when either the 5' or 3' neighbor was deoxyguanosine. The binding of (+)-anti-BPDE to oligodeoxyribonucleotide duplexes containing different numbers of consecutive dGs was analyzed in order to determine how the length of these sequences influences binding. Increases in the length of consecutive deoxyguanosine residues from 3 to 5 had little effect on the quantity of (+)-anti-BPDE bound to dG above that expected from the presence of a neighboring dG and an increase in the number of dG residues available for reaction. The results obtained with these oligodeoxyribonucleotide duplexes were consistent with the data available for the reaction of (+)-anti-BPDE with DNA, indicating that these duplexes are a valuable model for studying the effect of base sequence on the interaction of BPDE isomers with DNA. The dinucleotide postlabeling technique developed for these studies, with appropriate oligodeoxyribonucleotides and chromatographic conditions, will be useful for determining the effect of base sequence on the binding of other hydrocarbon diol epoxides as well as other reactive hydrocarbon metabolites to deoxyguanosine or deoxyadenosine in oligodeoxyribonucleotide duplexes and fragments of DNA.

Molecular cloning and sequencing of infC, the gene encoding translation initiation factor IF3, from four enterobacterial species.

Translation initiation factor IF3 plays a crucial role in initiation of protein synthesis in bacteria. In order to elucidate the IF3 structural elements required for these functions, the evolutionary conservation of IF3 and its gene, infC, was investigated. Homologous infC sequences from Salmonella typhimurium, Klebsiella pneumoniae, Serratia marcescens and Proteus vulgaris were amplified by the polymerase chain reaction and sequenced. Analysis of these sequences, as well as that from Bacillus stearothermophilus, revealed several regions (e.g. residues 62-73 and 173-177) of absolute sequence conservation, suggesting an important role for these regions in IF3 function.

Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans lesions.

Current laboratory diagnosis of Lyme disease relies on tests for the detection of antibodies to Borrelia burgdorferi, the etiologic agent of the disease. These tests are often unreliable because of a lack of sensitivity and specificity and test-to-test variability. The purpose of this study was to evaluate the sensitivity and specificity of polymerase chain reaction (PCR) amplification for detection of B. burgdorferi in skin biopsy specimens. Forty-six 2-mm skin biopsy samples were obtained from 44 patients with a clinical diagnosis of erythema migrans, 9 of whom were receiving antibiotic therapy at the time of biopsy. Specimens were ground in BSK medium with separate aliquots taken for culture and PCR. Of the specimens from the untreated group, 57% (21 of 37) were positive by culture and 22% (8 of 37) were culture negative; 22% (8 of 37) of the cultures were uninformative because of contamination. By comparison, 22 (59%) of 37 specimens were positive by PCR amplification. Of 21 culture-positive samples, 13 (62%) were also positive by PCR analysis. Thus, the sensitivity of the PCR was 59 to 62%, based on either a clinical or cultural diagnosis of untreated Lyme disease. None of the nine specimens from antibiotic-treated patients grew in culture, whereas two of the nine were positive by PCR analysis. Given the complexity and time required for culture, PCR is a promising technique for the diagnosis of early Lyme disease.