Discrete Structure of the Brain Rhythms.

Neuronal activity in the brain generates synchronous oscillations of the Local Field Potential (LFP). The traditional analyses of the LFPs are based on decomposing the signal into simpler components, such as sinusoidal harmonics. However, a common drawback of such methods is that the decomposition primitives are usually presumed from the onset, which may bias our understanding of the signal's structure. Here, we introduce an alternative approach that allows an impartial, high resolution, hands-off decomposition of the brain waves into a small number of discrete, frequency-modulated oscillatory processes, which we call oscillons. In particular, we demonstrate that mouse hippocampal LFP contain a single oscillon that occupies the θ-frequency band and a couple of γ-oscillons that correspond, respectively, to slow and fast γ-waves. Since the oscillons were identified empirically, they may represent the actual, physical structure of synchronous oscillations in neuronal ensembles, whereas Fourier-defined "brain waves" are nothing but poorly resolved oscillons.

Pretransplant IgG reactivity to apoptotic cells correlates with late kidney allograft loss.

Preexisting serum antibodies have long been associated with graft loss in transplant recipients. While most studies have focused on HLA-specific antibodies, the contribution of non-HLA-reactive antibodies has been largely overlooked. We have recently characterized mAbs secreted by B cell clones derived from kidney allograft recipients with rejection that bind to apoptotic cells. Here, we assessed the presence of such antibodies in pretransplant serum from 300 kidney transplant recipients and examined their contribution to the graft outcomes. Kaplan-Meier survival analysis revealed that patients with high pretransplant IgG reactivity to apoptotic cells had a significantly increased rate of late graft loss. The effect was only apparent after approximately 1 year posttransplant. Moreover, the association between pretransplant IgG reactivity to apoptotic cells and graft loss was still significant after excluding patients with high reactivity to HLA. This reactivity was almost exclusively mediated by IgG1 and IgG3 with complement fixing and activating properties. Overall, our findings support the view that IgG reactive to apoptotic cells contribute to presensitization. Taking these antibodies into consideration alongside anti-HLA antibodies during candidate evaluation would likely improve the transplant risk assessment.

Polyreactive antibodies developing amidst humoral rejection of human kidney grafts bind apoptotic cells and activate complement.

Antibody mediated rejection (AMR) is associated with a variety of graft-reactive antibodies following kidney transplant. To characterize these antibodies, we immortalized 107 B cell clones from a patient with AMR. In a previous study, we showed that six clones were reacting to multiple self-antigens as well as to HLA and MICA for two of them, thus displaying a pattern of polyreactivity. We show here that all six polyreactive clones also reacted to apoptotic but not viable cells. More generally we observed a nearly perfect overlap between polyreactivity and reactivity to apoptotic cells. Functionally, polyreactive antibodies can activate complement, resulting in the deposition of C3d and C4d at the surface of target cells. Testing the serum of 88 kidney transplant recipients revealed a significantly higher IgG reactivity to apoptotic cells in AMR patients than in patients with stable graft function. Moreover, total IgG purified from AMR patients had increased complement activating properties compared to IgG from non-AMR patients. Overall, our studies show the development of polyreactive antibodies cross-reactive to apoptotic cells during AMR. Further studies are now warranted to determine their contribution to the detection of C4d in graft biopsies as well as their role in the pathophysiology of AMR.

Expansion of polyreactive B cells cross-reactive to HLA and self in the blood of a patient with kidney graft rejection.

Antibody rejection is often accompanied by nondonor HLA specific antibodies (NDSA) and self-reactive antibodies that develop alongside donor-specific antibodies (DSA). To determine the source of these antibodies, we immortalized 107 B-cell clones from a kidney transplant recipient with humoral rejection. Two of these clones reacted to HLA class I or MICA. Both clones were also reactive to self-antigens and a lysate of a kidney cell line, hence revealing a pattern of polyreactivity. Monoclonality was verified by the identification of a single rearranged immunoglobulin heavy chain variable region (VH) sequence for each clone. By tracking their unique CDR3 sequence, we found that one such polyreactive clone was highly expanded in the patient blood, representing ~0.2% of circulating B cells. The VH sequence of this clone showed evidence of somatic mutations that were consistent with its memory phenotype and its expansion. Lastly, the reactivity of the expanded polyreactive B-cell clone was found in the patient serum at time of rejection. In conclusion, we provide here proof of principle at the clonal level that human antibodies can cross-react to HLA and self. Our findings strongly suggest that polyreactive antibodies contribute to DSA, NDSA as well as autoantibodies, in transplant recipients.

Chronic humoral rejection of human kidney allografts is associated with MMP-2 accumulation in podocytes and its release in the urine.

Chronic humoral rejection (CHR) is an important cause of late graft failures following kidney transplantation. Overall, the pathophysiology of CHR is poorly understood. Matrix metalloproteinase-2 (MMP-2), a type IV collagenase, has been implicated in chronic kidney disease and allograft rejection in previous studies. We examined the presence of MMP-2 in allograft biopsies and in the urine of kidney transplant recipients with CHR. MMP-2 staining was detected by immunohistochemistry in podocytes for all CHR patients but less frequently in patients with other renal complications. Urinary MMP-2 levels were also significantly higher in CHR patients (median 4942 pg/mL, N = 27) compared to non-CHR patients (median 598 pg/mL, N = 65; p < 0.001). Elevated urinary MMP-2 correlated with higher levels of proteinuria in both CHR and non-CHR patients. Longitudinal analysis indicated that increase in urine MMP-2 coincided with initial diagnosis of CHR as documented by the biopsies. Using an enzymatic assay, we demonstrated that MMP-2 was present in its active form in the urine of patients with CHR. Overall, our findings associate MMP-2 with glomerular injury as well as interstitial fibrosis and tubular atrophy observed in patients with CHR.

Site-directed mutagenesis of the katG gene of Mycobacterium tuberculosis: effects on catalase-peroxidase activities and isoniazid resistance.

Recent studies examining the molecular mechanisms of isoniazid (INH) resistance in Mycobacterium tuberculosis have demonstrated that a significant percentage of drug-resistant strains are mutated in the katG gene which encodes a catalase-peroxidase, and the majority of these alterations are missense mutations which result in the substitution of a single amino acid. In previous reports, residues which may be critical for enzymatic activity and the drug-resistant phenotype have been identified by evaluating INH-resistant clinical isolates and in vitro mutants. In this study, site-directed mutagenesis techniques were utilized to alter the wild-type katG gene from M. tuberculosis at 13 of these codons. The effects of these mutations were determined using complementation assays in katG-defective, INH-resistant strains of Mycobacterium smegmatis and Mycobacterium bovis BCG. This mutational analysis revealed that point mutations in the katG gene at nine of the 13 codons can cause drug resistance, and that enzymatic activity and resistance to INH are inversely related. In addition, mutations in the mycobacterial catalase-peroxidase which reduce catalase activity also decrease peroxidase activity.

Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda.

Transcription of downstream genes in the early operons of phage lambda requires a promoter-proximal element known as nut. This site acts in cis in the form of RNA to assemble a transcription antitermination complex which is composed of lambda N protein and at least four host factors. The nut-site RNA contains a small stem-loop structure called boxB. Here, we show that boxB RNA binds to N protein with high affinity and specificity. While N binding is confined to the 5' subdomain of the stem-loop, specific N recognition relies on both an intact stem-loop structure and two critical nucleotides in the pentamer loop. Substitutions of these nucleotides affect both N binding and antitermination. Remarkably, substitutions of other loop nucleotides also diminish antitermination in vivo, yet they have no detectable effect on N binding in vitro. These 3' loop mutants fail to support antitermination in a minimal system with RNA polymerase (RNAP), N, and the host factor NusA. Furthermore, the ability of NusA to stimulate the formation of the RNAP-boxB-N complex is diminished with these mutants. Hence, we suggest that boxB RNA performs two critical functions in antitermination. First, boxB binds to N and secures it near RNAP to enhance their interaction, presumably by increasing the local concentration of N. Second, boxB cooperates with NusA, most likely to bring N and RNAP in close contact and transform RNAP to the termination-resistant state.

Gynecological surgical outcomes among asymptomatic human immunodeficiency virus-infected women and uninfected control subjects.

To determine the effect of asymptomatic human immunodeficiency virus (HIV) infection on the risk of complications and outcomes in women undergoing gynecological surgical procedures, retrospective analysis was performed of 62 asymptomatic HIV-infected women who underwent gynecological procedures. One hundred forty seronegative women who had similar procedures during the same time period served as controls. Procedures included tubal sterilization, hysterectomy, and diagnostic laparotomy. The following variables were compared: length of hospital stay, age, blood loss, white blood cell count, hemoglobin, and hematocrit. Laboratory parameters were compared pre-and postoperatively, as well as between the study and control groups. Race and parity were similar in both groups. HIV-infected women were younger (mean: 25 years versus 31 years) than controls. Length of hospital stay was similar. Blood loss was higher in the HIV-infected group than controls. (318 cc versus 122 cc) Differences in white blood cell counts, hematocrits, and febrile morbidity were insignificant. Asymptomatic HIV infection has minimal effect on the outcome of elective gynecologic surgery. The younger age of the HIV-infected women reflects the demographics of HIV infection and sterilization reflects the desire to prevent perinatal transmission.

Mental health under national health care reform: the empirical foundations.

This article reviews research pertinent to mental health services under several U.S. health care reform proposals. Issues examined include the redistributional impact of the inclusion of outpatient mental health benefits, optimal benefit packages, and findings that mental health services lower medical utilization costs. It is argued that extending a minimalist model of time-limited benefits, similar to that implemented in most managed care programs, to a national health care insurance plan would perpetuate the current two-class mental health care system.

Oligodendrocyte precursor quantitation and localization in perinatal brain using a retrospective bioassay.

Several late stages of the oligodendrocyte (OL) developmental lineage can be identified immunologically in the newborn rat brain. However, OL lineage-specific markers are not available for the detection of the less mature, yet determined, OL precursors. We have developed a retrospective bioassay, combining limiting dilution analysis with a novel culture system, that quantitatively assesses the developmental potential in vivo of phenotypically undefined OL precursors in order to (1) demonstrate their existence, (2) estimate their total number in the premyelinated rat brain, and (3) demonstrate their presence in regions distal to germinal zones at times previously predicted to be devoid of such cells. Between embryonic day (E) 21 and postnatal day (P) 0, cells determined to become oligodendrocytes increase in frequency approximately 5-fold in the whole brain (from one precursor for every 365 cells to 1 in 74), and approximately 2.5-fold in the telencephalon (from 1 in 298 to 1 in 115). From these data it is calculated that a pool of approximately 10(6) phenotypically undefined cells are present in the newborn brain that are able to differentiate into OL in vitro. Further, by applying this assay to tissue samples of subdomains of the developing cerebellum, we have demonstrated that such cells are present in large numbers as early as E20 in regions sparsely populated with cells expressing the blastic neural cell marker ganglioside GD3, suggesting that they migrated to this position as a pre-GD3-expressing cell. These results significantly change the predicted ontogeny of the oligodendrocyte lineage and should fuel the ongoing search for these early OL precursors.

Control of transcription processivity in phage lambda: Nus factors strengthen the termination-resistant state of RNA polymerase induced by N antiterminator.

During transcription of phage lambda early operons, the N gene product alters host RNA polymerase (RNAP) so that transcription proceeds through multiple stop signals. Here, we reproduce the essence of N activity with purified components in synthetic transcription units that contain lambda pL promoter and the N-recognition site, nutL, followed by a variety of intrinsic terminators. We show that three host factors (NusA, NusE, and NusG) are essential for N to allow appreciable transcription through multiple terminators and that this persistent antitermination is stimulated by a fourth factor, NusB. Remarkably, in the absence of all four factors, N suppresses various terminators placed near the nut site. This basal antitermination activity of N is enhanced by NusA and is diminished by high salt and temperature. We postulate that N interacts with RNAP directly, inducing the termination-resistant state. While NusA facilitates this interaction, the other factors strengthen it sufficiently over time and distance so that RNAP bypasses multiple terminators. The dispensability of NusB for persistent antitermination in vitro, but not in vivo, raises the possibility that NusB performs two functions: it increases the stability of N antitermination complex and also counteracts an inhibitory factor in the cell.

Bioequivalence of oral and injectable levoleucovorin and leucovorin.

The bioequivalence and bioavailability of oral and intravenous formulations of levoleucovorin and leucovorin were studied, and the absolute bioavailabilities of levoleucovorin and leucovorin tablet formulations were determined. Healthy male volunteers participated in two randomized, single-dose, four-way crossover studies. The treatment groups were as follows: A = five 2.5-mg levoleucovorin tablets, B = five 5-mg leucovorin tablets, C = one 12.5-mg levoleucovorin tablet, D = one 25-mg leucovorin tablet, in study 1; A = 15-mg levoleucovorin injection, B = two 7.5-mg levoleucovorin tablets, C = 30-mg leucovorin injection, D = two 15-mg leucovorin tablets, in study 2. Serum concentrations of N-5-methyltetrahydrofolate (the primary metabolite and circulating form of reduced folate after leucovorin administration) and total tetrahydrofolate were measured over 24 hours after dose administration. Pharmacokinetic values were calculated for N-5-methyltetrahydrofolate and total tetrahydrofolates; values were compared for A versus B, C versus D, A versus C, and B versus D in study 1 and A versus C and B versus D in study 2. Results from 35 men in study 1 and 33 in study 2 showed that 12.5-mg oral doses and 15-mg intravenous doses of levoleucovorin are bioequivalent to 25-mg oral doses and 30-mg intravenous doses of leucovorin, respectively. Equivalence was observed after oral and intravenous administration. The absolute bioavailability of levoleucovorin (74%) was not significantly different from that of leucovorin (65%).(ABSTRACT TRUNCATED AT 250 WORDS)

Specificity of antitermination mechanisms. Suppression of the terminator cluster T1-T2 of Escherichia coli ribosomal RNA operon, rrnB, by phage lambda antiterminators.

Transcription of the ribosomal RNA operons (rrn) in Escherichia coli is subject to an antitermination mechanism whereby RNA polymerase is modified to a termination-resistant form during transit through the rrn leader region. This antitermination mechanism is unable to overcome the T1-T2 terminator cluster located at the end of an rrn operon, such as rrnB. We have tested the specificity with which the T1-T2 terminators override an antitermination mechanism, by placing the terminator cluster downstream from the nut and qut sites recognized by phage lambda N and Q gene antiterminators, respectively. Measurement of downstream gene expression shows that RNA polymerase modified by either N or Q reads through the T1-T2 terminators quite efficiently. This supports the view that T1-T2 are not superterminators, and that the rrn antitermination mechanism may have a restricted terminator specificity.

Phase I/pharmacokinetic reevaluation of thioTEPA.

Because the initial evaluation of N,N',N''-triethylenethiophosphoramide (thioTEPA) preceded the standardized approach to the Phase I trials, uncertainty surrounds the recommended dose. Since it has recently been demonstrated that an almost 100-fold increase in dose can be administered in bone marrow transplant regimens, we conducted a Phase I reevaluation of thioTEPA. ThioTEPA was administered i.v. in 50 ml 5% dextrose in water over 10 min. Twenty-seven patients were entered at doses ranging from 30 to 75 mg/m2. The major toxic effect was myelosuppression; thrombocytopenia greater than or equal to grade 3 occurred in four of seven patients, and leukopenia greater than or equal to grade 3 in two of seven patients at 75 mg/m2. Among eight patients at 65 mg/m2 only two had greater than or equal to grade 3 myelosuppression making this the recommended new phase II dose for the majority of patients. Moderate (grade 2) easily controlled nausea and vomiting was the only other major side effect. There was no alopecia or mucosal or neurological toxicity. Three partial remissions were observed among nine previously treated ovarian cancer patients. Plasma concentrations of thioTEPA and its major active metabolite triethylenephosphoramide (TEPA) were measured by gas chromatography. The half-life of thioTEPA ranged from 51.6 to 211.8 min, and its pharmacokinetics was dose dependent; total body thioTEPA clearance decreased with increasing dose. The half-life of TEPA was considerably longer than that of the parent compound (3.0 to 21.1 h); as a result, the area under the plasma concentration-time curve (AUC) of TEPA was severalfold greater than that of the parent compound. The ratio of TEPA AUC to thioTEPA AUC decreased with increasing dose, suggesting that formation of TEPA is a saturable step in elimination. The AUC and total body clearance of thioTEPA, but not of TEPA, were closely correlated with neutrophil but not platelet toxicity.

Neurons controlling cardiovascular responses to emotion are located in lateral hypothalamus-perifornical region.

We did four experiments to determine whether the lateral hypothalamus-perifornical (LH/PF) region is the source of neuronal cell bodies responsible for producing the cardiovascular (CV) responses associated with emotion or the defense reaction. Of particular concern was whether the paraventricular nucleus (PVN) plays a role in the generation of these CV responses. Mapping the hypothalamus with electrical stimulation showed that the CV pattern of responses was never produced by stimulating the PVN and was invariably produced by stimulating the LH/PF region. Complete electrolytic destruction of the PVN and subsequent axonal degeneration did not change the CV pattern of responses elicited by LH/PF stimulation, whereas any encroachment of the lesion on the LH/PF region decreased the magnitude of the CV responses. Injection of the neuroexcitotoxin ibotenic acid (Ibo) into the PVN did not affect responses to LH/PF stimulation, whereas Ibo injection into the LH/PF region eliminated or severely attenuated the CV responses. Retrograde labeling of cells from the thoracic cord and the ventrolateral reticular formation revealed a scattered group of cells in the LH/PF region that may be the cells controlling the CV responses. These results point directly to the LH/PF region as the source of the cell bodies responsible for the autonomic responses associated with emotion or defense reactions.

Volumetric growth of the major brain divisions in fetal Macaca nemestrina.

Brains from 22 age-dated fetal Macaca nemestrina were embedded in celloidin and prepared for histological serial sections. A computer-based morphometric system was used to digitize contours of neural structures and to calculate their areas and volumes. Shrinkage of the brain sections was corrected by a multiplication factor relating pre-processed brain volume to the computer-calculated volume of the processed brain. Volume growth of the telencephalon, mesencephalon and pons-medulla was linear over the fetal period of 60 days postconception to near-term at 166 days postconception. Volume growth of the total brain, diencephalon and cerebellum, was curvilinear with respect to age, with slower growth initially and faster growth in the later stages of gestation. Total brain and body grew with an almost 1:1 relation during the fetal period. The proportionate growth of the brain was largely accounted for by telencephalic growth. The other brain divisions all showed different growth rates in relation to growth of the body.

Lack of apparent effect of assay methodology on the pharmacokinetics of digoxin.

The purpose of this study was to determine if serum digoxin concentration data using three different automated immunoassay methods would produce similar pharmacokinetic values in normal volunteer subjects. Area under the curve (AUC), steady-state volume of distribution/bioavailability ratio (Vd/F), terminal elimination rate constant (beta), clearance/bioavailability ratio (CL/F), maximum digoxin concentration (Cmax), minimum digoxin concentration (Cmin), and time of peak (Tp) were evaluated. Ten healthy volunteers received digoxin capsules 0.2 mg daily for 10 days. On day 10, 16 serial blood samples were collected over a 24-h dosing interval and analyzed by radioimmunoassay (RIA) (Concept 4, Micromedic Systems), fluorescence polarization immunoassay (FPIA) (TDx, Abbott Laboratories), and affinity column-mediated immunoassay (ACMIA), (aca, duPont Instruments). When comparing RIA and FPIA, the mean of the percent differences for AUC, Vd/F, beta, and CL/F were 9, 4, 10, and 6%, respectively. The mean of the percent differences were 2, 3, 44, and 6%, respectively, when comparing RIA and ACMIA. However, none of these differences were statistically significant. Although a trend toward higher Cmax values by RIA was noted, there was no statistical difference in Cmax, Cmin, and Tp. Orthogonal regression of all serum digoxin concentrations showed that FPIA = 0.76 RIA + 0.19, r = 0.967 (p less than 0.001); and ACMIA = 0.92 RIA + 0.04, r = 0.943 (p less than 0.001). At serum digoxin concentrations less than 1 ng/ml, FPIA overestimated RIA results (p less than 0.005), while ACMIA was approximately equal to the RIA results.(ABSTRACT TRUNCATED AT 250 WORDS)

Oral ciprofloxacin in the treatment of serious soft tissue and bone infections. Efficacy, safety, and pharmacokinetics.

Forty-eight patients were enrolled in a clinical study of oral ciprofloxacin for the treatment of soft tissue or bone infections. Patients received 500 to 750 mg of ciprofloxacin every 12 hours. In the predominantly older population studied, there were 13 patients with osteomyelitis, 24 diabetic patients with soft tissue infection and probable osteomyelitis, and 11 patients with other soft tissue infections. Infecting pathogens included Pseudomonas aeruginosa in 25 patients, Serratia species in nine patients, Staphylococcus aureus in 13 patients, and other aerobic gram-negative rods in 21 patients. Clinical response (defined as resolution or improvement) was noted in 84 percent of patients with non-diabetic osteomyelitis, in 79 percent of patients with diabetic infections, and in 91 percent of patients with soft tissue infections. Microbiologic outcome was very favorable in 75 percent of cases, and Pseudomonas responded as well as any other pathogen. Pharmacokinetic properties of ciprofloxacin were evaluated in 12 patients, and the data were analyzed using both compartmental and non-compartmental analyses. Mean values for compartmental rate constants (hours-1) were as follows: absorption rate constant = 1.15; intercompartmental rate constants, k12 = 0.48, and k21 = 0.58; elimination rate constant = 0.46; distribution rate constant = 1.31; and terminal elimination rate constant = 0.19. The apparent volume of distribution at steady state/bioavailability was 196 liters and total body clearance/bioavailability was 45.9 liters/hour. The mean time to peak concentration was 1.3 hours. The mean peak concentration as determined by compartmental fitting (2.4 micrograms/ml) underestimated the observed peak (3.2 micrograms/ml) by 24.8 percent. Clearance of ciprofloxacin was similar regardless of the method used to fit the data, whereas the volume of distribution was significantly different when the two analysis techniques were compared. Ciprofloxacin was well tolerated, with the most frequent adverse reactions being rash, gastrointestinal intolerance, and increased levels of liver enzymes, each of which occurred in five patients.