Demographic and Geographic Characterization of Excess Mortality During the COVID-19 Pandemic in Baltimore City, Maryland, March 2020 to March 2021.

Estimates of excess mortality can provide insight into direct and indirect impacts of the coronavirus disease 2019 (COVID-19) pandemic beyond deaths specifically attributed to COVID-19. We analyzed death certificate data from Baltimore City, Maryland, from March 1, 2020, to March 31, 2021, and found that 1,725 individuals (95% confidence interval: 1,495, 1,954) died in excess of what was expected from all-cause mortality trends in 2016-2019; 1,050 (61%) excess deaths were attributed to COVID-19. Observed mortality was 23%-32% higher than expected among individuals aged 50 years and older. Non-White residents of Baltimore City also experienced 2 to 3 times higher rates of excess mortality than White residents (e.g., 37.4 vs. 10.7 excess deaths per 10,000 population among Black residents vs. White residents). There was little to no observed excess mortality among residents of hospice, long-term care, and nursing home facilities, despite accounting for nearly 30% (312/1,050) of recorded COVID-19 deaths. There was significant geographic variation in excess mortality within the city, largely following racial population distributions. These results demonstrate the substantial and unequal impact of the COVID-19 pandemic on Baltimore City residents and the importance of building robust, timely surveillance systems to track disparities and inform targeted strategies to remediate the impact of future epidemics.

Fine root dynamics across pantropical rainforest ecosystems.

Fine roots constitute a significant component of the net primary productivity (NPP) of forest ecosystems but are much less studied than aboveground NPP. Comparisons across sites and regions are also hampered by inconsistent methodologies, especially in tropical areas. Here, we present a novel dataset of fine root biomass, productivity, residence time, and allocation in tropical old-growth rainforest sites worldwide, measured using consistent methods, and examine how these variables are related to consistently determined soil and climatic characteristics. Our pantropical dataset spans intensive monitoring plots in lowland (wet, semi-deciduous, and deciduous) and montane tropical forests in South America, Africa, and Southeast Asia (n = 47). Large spatial variation in fine root dynamics was observed across montane and lowland forest types. In lowland forests, we found a strong positive linear relationship between fine root productivity and sand content, this relationship was even stronger when we considered the fractional allocation of total NPP to fine roots, demonstrating that understanding allocation adds explanatory power to understanding fine root productivity and total NPP. Fine root residence time was a function of multiple factors: soil sand content, soil pH, and maximum water deficit, with longest residence times in acidic, sandy, and water-stressed soils. In tropical montane forests, on the other hand, a different set of relationships prevailed, highlighting the very different nature of montane and lowland forest biomes. Root productivity was a strong positive linear function of mean annual temperature, root residence time was a strong positive function of soil nitrogen content in montane forests, and lastly decreasing soil P content increased allocation of productivity to fine roots. In contrast to the lowlands, environmental conditions were a better predictor for fine root productivity than for fractional allocation of total NPP to fine roots, suggesting that root productivity is a particularly strong driver of NPP allocation in tropical mountain regions.

Wood construction more strongly shapes deadwood microbial communities than spatial location over 5 years of decay.

Diverse communities of fungi and bacteria in deadwood mediate wood decay. While rates of decomposition vary greatly among woody species and spatially distinct habitats, the relative importance of these factors in structuring microbial communities and whether these shift over time remains largely unknown. We characterized fungal and bacterial diversity within pieces of deadwood that experienced 6.3-98.8% mass loss while decaying in common garden 'rotplots' in a temperate oak-hickory forest in the Ozark Highlands, MO, USA. Communities were isolated from 21 woody species that had been decomposing for 1-5 years in spatially distinct habitats at the landscape scale (top and bottom of watersheds) and within stems (top and bottom of stems). Microbial community structure varied more strongly with wood traits than with spatial locations, mirroring the relative role of these factors on decay rates on the same pieces of wood even after 5 years. Co-occurring fungal and bacterial communities persistently influenced one another independently from their shared environmental conditions. However, the relative influence of wood construction versus spatial locations differed between fungi and bacteria, suggesting that life history characteristics of these clades structure diversity differently across space and time in decomposing wood.

Accurate forest projections require long-term wood decay experiments because plant trait effects change through time.

Whether global change will drive changing forests from net carbon (C) sinks to sources relates to how quickly deadwood decomposes. Because complete wood mineralization takes years, most experiments focus on how traits, environments and decomposer communities interact as wood decay begins. Few experiments last long enough to test whether drivers change with decay rates through time, with unknown consequences for scaling short-term results up to long-term forest ecosystem projections. Using a 7 year experiment that captured complete mineralization among 21 temperate tree species, we demonstrate that trait effects fade with advancing decay. However, wood density and vessel diameter, which may influence permeability, control how decay rates change through time. Denser wood loses mass more slowly at first but more quickly with advancing decay, which resolves ambiguity about the after-life consequences of this key plant functional trait by demonstrating that its effect on decay depends on experiment duration and sampling frequency. Only long-term data and a time-varying model yielded accurate predictions of both mass loss in a concurrent experiment and naturally recruited deadwood structure in a 32-year-old forest plot. Given the importance of forests in the carbon cycle, and the pivotal role for wood decay, accurate ecosystem projections are critical and they require experiments that go beyond enumerating potential mechanisms by identifying the temporal scale for their effects.

Uptake of HPV testing and extended cervical cancer screening intervals following cytology alone and Pap/HPV cotesting in women aged 30-65 years.

OBJECTIVE: To evaluate the adoption of HPV testing and recommended extended cervical cancer screening intervals in clinical practice, we described yearly uptake of Pap/HPV cotesting and estimated length of time between normal screens by patient characteristics. METHODS: We examined 55,575 Pap/HPV records from 27,035 women aged 30-65 years from the Johns Hopkins Hospital Pathology Data System between 2006 and 2013. Cotest uptake and median times to next screening test for cotests and cytology only were calculated. Adjusted hazard ratios were estimated using Cox proportional hazards models, with random effects adjustment for clustering within clinic. RESULTS: Cotest usage increased from < 10% in 2006 to 78% in 2013. The median time to next screening test following normal cytology alone remained constant around 1.5 years. Screening intervals following a dual-negative cotest increased from 1.5 years in 2006/2007 to 2.5 years in 2010, coincident with increases in the proportion of women cotested. Intervals following a dual negative cotest were longer among Medicare patients (3 years) compared with privately insured women (2.5 years), and shorter among black (2 years) compared with white women (2.8 years). CONCLUSION: By mid-2013 we observed broad adoption of Pap/HPV cotesting in routine screening in a large academic medical center. Increased screening intervals were observed only among cotested women, while those screened by cytology alone continued to be screened almost annually. The influence of different combinations of race and insurance on screening intervals should be further evaluated to ensure balance of screening risks and benefits in the U.S.

Effect of patient navigation on colorectal cancer screening in a community-based randomized controlled trial of urban African American adults.

PURPOSE: In recent years, colorectal cancer (CRC) screening rates have increased steadily in the USA, though racial and ethnic disparities persist. In a community-based randomized controlled trial, we investigated the effect of patient navigation on increasing CRC screening adherence among older African Americans. METHODS: Participants in the Cancer Prevention and Treatment Demonstration were randomized to either the control group, receiving only printed educational materials (PEM), or the intervention arm where they were assigned a patient navigator in addition to PEM. Navigators assisted participants with identifying and overcoming screening barriers. Logistic regression analyses were used to assess the effect of patient navigation on CRC screening adherence. Up-to-date with screening was defined as self-reported receipt of colonoscopy/sigmoidoscopy in the previous 10 years or fecal occult blood testing (FOBT) in the year prior to the exit interview. RESULTS: Compared with controls, the intervention group was more likely to report being up-to-date with CRC screening at the exit interview (OR 1.55, 95 % CI 1.07-2.23), after adjusting for select demographics. When examining the screening modalities separately, the patient navigator increased screening for colonoscopy/sigmoidoscopy (OR 1.53, 95 % CI 1.07-2.19), but not FOBT screening. Analyses of moderation revealed stronger effects of navigation among participants 65-69 years and those with an adequate health literacy level. CONCLUSIONS: In a population of older African Americans adults, patient navigation was effective in increasing the likelihood of CRC screening. However, more intensive navigation may be necessary for adults over 70 years and individuals with low literacy levels.

Herbivory makes major contributions to ecosystem carbon and nutrient cycling in tropical forests.

The functional role of herbivores in tropical rainforests remains poorly understood. We quantified the magnitude of, and underlying controls on, carbon, nitrogen and phosphorus cycled by invertebrate herbivory along a 2800 m elevational gradient in the tropical Andes spanning 12°C mean annual temperature. We find, firstly, that leaf area loss is greater at warmer sites with lower foliar phosphorus, and secondly, that the estimated herbivore-mediated flux of foliar nitrogen and phosphorus from plants to soil via leaf area loss is similar to, or greater than, other major sources of these nutrients in tropical forests. Finally, we estimate that herbivores consume a significant portion of plant carbon, potentially causing major shifts in the pattern of plant and soil carbon cycling. We conclude that future shifts in herbivore abundance and activity as a result of environmental change could have major impacts on soil fertility and ecosystem carbon sequestration in tropical forests.

Qualitative and quantitative evaluation of human dental enamel after bracket debonding: a noncontact three-dimensional optical profilometry analysis.

OBJECTIVES: The aim of this study was to undertake a qualitative and quantitative evaluation of changes on enamel surfaces after debonding of brackets followed by finishing procedures, using a high-resolution three-dimensional optical profiler and to investigate the accuracy of the technique. MATERIALS AND METHODS: The labial surfaces of 36 extracted upper central incisors were examined. Before bonding, the enamel surfaces were subjected to profilometry, recording four amplitude parameters. Brackets were then bonded using two types of light-cured orthodontic adhesive: composite resin and resin-modified glass ionomer cement. Finishing was performed by three different methods: pumice on a rubber cup, fine and ultrafine aluminum oxide discs, and microfine diamond cups followed by silicon carbide brushes. The samples were subsequently re-analyzed by profilometry. RESULTS: Wilcoxon signed-rank test, Kruskal-Wallis test (p < 0.05) and a posteriori Mann-Whitney U test with Bonferroni correction (p < 0.0167) revealed a significant reduction of enamel roughness when diamond cups followed by silicon carbide brushes were used to finish surfaces that had remnants of resin-modified glass ionomer adhesive and when pumice was used to finish surfaces that had traces of composite resin. Enamel loss was minimal. CONCLUSIONS: The 3D optical profilometry technique was able to provide accurate qualitative and quantitative assessment of changes on the enamel surface after debonding. CLINICAL RELEVANCE: Morphological changes in the topography of dental surfaces, especially if related to enamel loss and roughness, are of considerable clinical importance. The quantitative evaluation method used herein enables a more comprehensive understanding of the effects of orthodontic bonding on teeth.

The impact of insect herbivory on biogeochemical cycling in broadleaved forests varies with temperature.

Herbivorous insects alter biogeochemical cycling within forests, but the magnitude of these impacts, their global variation, and drivers of this variation remain poorly understood. To address this knowledge gap and help improve biogeochemical models, we established a global network of 74 plots within 40 mature, undisturbed broadleaved forests. We analyzed freshly senesced and green leaves for carbon, nitrogen, phosphorus and silica concentrations, foliar production and herbivory, and stand-level nutrient fluxes. We show more nutrient release by insect herbivores at non-outbreak levels in tropical forests than temperate and boreal forests, that these fluxes increase strongly with mean annual temperature, and that they exceed atmospheric deposition inputs in some localities. Thus, background levels of insect herbivory are sufficiently large to both alter ecosystem element cycling and influence terrestrial carbon cycling. Further, climate can affect interactions between natural populations of plants and herbivores with important consequences for global biogeochemical cycles across broadleaved forests.

Determinants of newly detected human papillomavirus infection in HIV-infected and HIV-uninfected injection drug using women.

BACKGROUND: We sought to identify factors associated with newly detected human papillomavirus (HPV) infection in a high-risk cohort of injection drug using women in Baltimore, MD. METHODS: We studied 146 HIV-infected and 73 HIV-uninfected female participants in a 5-year prospective HIV natural history study. We examined the association of sexual and nonsexual risk factors and newly detected type-specific HPV infection as determined by consensus PCR between consecutive visits. RESULTS: Newly detected HPV was more common among HIV-infected versus HIV-uninfected women (30% and 6%, respectively; P <0.01). Among the entire cohort, recent crack use (OR, 1.7; 95% CI, 1.1-2.6) and HIV infection/CD4 cell count were independent predictors for new HPV detection (HIV-uninfected as reference, OR, 4.6; 95% CI, 2.3-8.9, OR, 5.4; 95% CI, 2.8-10.3, and OR, 10.9; 95% CI, 5.5-21.7 for HIV-infected CD4 >500, 200-500, and <200, respectively). Among HIV-uninfected women, recent marijuana use was an independent predictor of newly detected HPV infection (OR, 3.5; 95% CI, 1.3-9.5). CONCLUSIONS: Newly detected HPV clearly increased with greater immunosuppression in HIV-infected injection drug users. Larger studies of HIV-uninfected and infected high-risk individuals are needed to clarify the independent associations of crack and marijuana use with new (or reactivated) HPV infection.

Evaluating the perioperative safety of laparoscopic radical nephrectomy for large, non-metastatic renal tumours: a comparative analysis of T1-T2 with T3a tumours.

OBJECTIVE: With increasing surgeon experience, the use of laparoscopic radical nephrectomy (LRN) in large and locally advanced renal tumours (T3a) is gaining favour in urological practice. There are limited studies reporting surgical outcomes in such groups. The aim of this study was to review our experience with LRN in these patients. METHODS: Data was retrospectively collected on 201 consecutive patients who underwent LRN for renal cancer by a single surgeon. Perioperative parameters assessed were age, gender, American Society of Anaesthesiologists score (ASA), waist circumference, tumour size, specimen size, histological subtypes, anaesthetic duration, operative approach and technique, surgery duration, blood loss, pre and postoperative renal function, complication rate and duration of hospital stay. RESULTS: Of 201 patients undergoing LRN, 43 (21%) patients had T3a tumours (group 2). The remaining 158 (79%) patients had T1 tumours (group1). Mean tumour size in group 2 was 12.2 cm. Renal cell carcinoma (RCC) was more common in males than females (131/201; 65%). Patients with T3a disease were more likely to have an ASA score of 2 (37/201; 18%). In the majority of patients across both groups, LRN was completed using a 3-port approach (173/201; 86%). There were no significant differences between groups in terms of mean anaesthetic duration, average surgical time, average estimated blood loss, complication rate and mean hospital stay. CONCLUSION: Our study shows that LRN has equivalent perioperative outcomes and safety in larger and locally advanced renal tumours.

Tetramerization of the LexA repressor in solution: implications for gene regulation of the E.coli SOS system at acidic pH.

Structural changes on LexA repressor promoted by acidic pH have been investigated. Intense protein aggregation occurred around pH 4.0 but was not detected at pH values lower than pH 3.5. The center of spectral mass of the Trp increased 400 cm(-1) at pH 2.5 relatively to pH 7.2, an indication that LexA has undergone structural reorganization but not denaturation. The Trp fluorescence polarization of LexA at pH 2.5 indicated that its hydrodynamic volume was larger than its dimer at pH 7.2. 4,4'-Dianilino-1,1'-binaphthyl-5,5'- disulfonic acid (bis-ANS) experiments suggested that the residues in the hydrophobic clefts already present at the LexA structure at neutral pH had higher affinity to it at pH 2.5. A 100 kDa band corresponding to a tetramer was obtained when LexA was subject to pore-limiting native polyacrylamide gel electrophoresis at this pH. The existence of this tetrameric state was also confirmed by small angle X-ray scattering (SAXS) analysis at pH 2.5. 1D 1H NMR experiments suggested that it was composed of a mixture of folded and unfolded regions. Although 14,000-fold less stable than the dimeric LexA, it showed a tetramer-monomer dissociation at pH 2.5 from the hydrostatic pressure and urea curves. Albeit with half of the affinity obtained at pH 7.2 (Kaff of 170 nM), tetrameric LexA remained capable of binding recA operator sequence at pH 2.5. Moreover, different from the absence of binding to the negative control polyGC at neutral pH, LexA bound to this sequence with a Kaff value of 1415 nM at pH 2.5. A binding stoichiometry experiment at both pH 7.2 and pH 2.5 showed a [monomeric LexA]/[recA operator] ratio of 2:1. These results are discussed in relation to the activation of the Escherichia coli SOS regulon in response to environmental conditions resulting in acidic intracellular pH. Furthermore, oligomerization of LexA is proposed to be a possible regulation mechanism of this regulon.

Predictors of Human Papillomavirus Vaccination in a Large Clinical Population of Males Aged 11 to 26 years in Maryland, 2012-2013.

BACKGROUND: Despite the recommendation for routine human papillomavirus (HPV) vaccination in males, coverage estimates remain low. We sought to identify predictors of receiving each HPV vaccine dose among a large clinical population of males. METHODS: We conducted a cross-sectional analysis of electronic medical records for 14,688 males ages 11 to 26 years attending 26 outpatient clinics (January 2012-April 2013) in Maryland to identify predictors of each HPV vaccine dose using multivariate logistic regression models with generalized estimating equations. All analyses were stratified in accordance with vaccine age recommendations: 11 to 12 years, 13 to 21 years, and 22 to 26 years. Analyses of predictors of receipt of subsequent HPV doses were also stratified by the number of clinic visits (≤3 and >3). RESULTS: Approximately 15% of males initiated the HPV vaccine. Less than half of males eligible received the second and third doses, 49% and 47%, respectively. Non-Hispanic black males (vs. non-Hispanic white) ages 11 to 12 and 13 to 21 years and males with public insurance (vs. private) ages 13 to 21 years had significantly greater odds of vaccine initiation, but significantly decreased odds of receiving subsequent doses, respectively. Attendance to >3 clinic visits attenuated the inverse association between public insurance and receipt of subsequent doses. CONCLUSION: Overall, rates of HPV vaccine initiation and of subsequent doses were low. While non-Hispanic black and publicly insured males were more likely to initiate the HPV vaccine, they were less likely to receive subsequent doses. IMPACT: Tailoring different intervention strategies for increasing HPV vaccine initiation versus increasing rates of subsequent doses among males may be warranted.

Antigenicity and immunogenicity of P30-derived peptides in experimental models of toxoplasmosis.

P30, also referred to as SAG-1, is now recognized as a major Toxoplasma gondii antigen potentially important for both diagnosis and immunoprophylaxis of toxoplasmosis. By using predictive algorithms, five synthetic peptides (48-67, 82-102, 213-230, 238-256 and 279-285) derived from P30, were investigated for B- and T-cell determinants in mouse and rat experimental models. Antibody recognition appeared more broadly distributed along the P30 sequence, whereas T-cell recognition was mainly targeted on the 238-256 peptide. In the absence of any carrier protein, this peptide induced a B- and T-cell immune response independent of the route of immunization (oral route or subcutaneous injection). This peptide (238-256) induced multiple antibody isotypes. In contrast with the 238-256 peptide, the 48-67 peptide, either free or in the form of a multiple antigenic peptide (MAP) construct or the 279-295 peptide, elicited antibodies associated with a TH2 response. This study reports for the first time the analysis of the antigenic and immunogenic properties of P30-derived peptides and are potentially useful for vaccinal strategies incorporating the P30 Toxoplasma gondii antigen.

The Relationship Between Education and Prostate-Specific Antigen Testing Among Urban African American Medicare Beneficiaries.

PURPOSE: We examined the association between socioeconomic status (SES) and prostate-specific antigen (PSA) cancer screening among older African American men. METHODS: We analyzed baseline data from a sample of 485 community-dwelling African American men who participated in the Cancer Prevention and Treatment Demonstration Trial. The outcome was receipt of PSA screening within the past year. SES was measured using income and educational attainment. Sequential multivariate logistic regression models were performed to study whether health care access, patient-provider relationship, and cancer fatalism mediated the relationship between SES and PSA screening. RESULTS: Higher educational attainment was significantly associated with higher odds of PSA screening in the past year (odds ratio (OR) 2.08 for college graduate compared to less than high school graduate, 95 % confidence interval (CI) 1.03-4.24); income was not. Health care access and patient-provider communication did not alter the relationship between education and screening; however, beliefs regarding cancer fatalism partially mediated the observed relationship. CONCLUSION: Rates of prostate cancer screening among African American men vary by level of educational attainment; beliefs concerning cancer fatalism help explain this gradient. Understanding the determinants of cancer fatalism is a critical next step in building interventions that seek to ensure equitable access to prostate cancer screening.

1,25 Dihydroxyvitamin D3 exerts regional effects in the central nervous system during experimental allergic encephalomyelitis.

1,25-dihydroxyvitamin D3 (1,25-D3) is already known to prevent clinical signs of experimental allergic encephalomyelitis when animals are treated during the immunization phase. In the present work we have evaluated the ability of 1,25-D3 to inhibit chronic relapsing experimental allergic encephalomylitis (EAE) of the Lewis rat, when administered after the beginning of clinical signs. We observed a significant clinical improvement in 1,25-D3-treated rats. This effect was accompanied by a profound inhibition of CD4 antigen expression by central nervous system (CNS) infiltrating monocytes/macrophages and parenchymal microglia. In addition, immunohistochemical analysis performed at the time of the second attack evidenced a region-specific distribution of inflammatory cells. In the same way, some aspects of the effects exerted by 1,25-D3 appeared to vary depending on the region considered, namely spinal cord, brainstem, cerebellum, midbrain or anterior brain. Thus, in 1,25-D3-treated rats, we observed an almost complete inhibition of CD4 antigen expression in the granule cell layer and the adjacent white matter of the cerebellum as well as a marked decrease in the number of OX42-positive cells (macrophages and activated microglia) in anterior brain sections. We conclude that 1,25-D3 can exert immunomodulatory effects inside the CNS during an ongoing immune process and may thus represent a promising therapy for multiple sclerosis.

Molecular characterization of a dense granule antigen (Gra 2) associated with the network of the parasitophorous vacuole in Toxoplasma gondii.

The monoclonal antibody (mAb) TG17.179 recognizes an excreted-secreted antigen (ESA) of 28.5 kDa named Gra 2, which is stored in the dense granules of Toxoplasma cells and secreted into the parasitophorous vacuole after host cell invasion. Screening of an expression cDNA library with TG17.179 led to the isolation of several clones, the longest one (clone L) being of 1030 bp. Clone L cDNA was found to be homologous to a previously described composite cDNA encoding a P28 protein of Toxoplasma gondii. Characterization of one genomic clone indicates that the complete GRA 2 gene is about 1.3 kb in length, including an intron of 241 bp. Northern blot and primer extension analyses confirmed the size of the mature messenger (1.1 kb). Amino acid partial sequencing of the native antigen purified by HPLC and metabolic radiolabelings of ESAs perfectly matched the primary amino acid structure deduced from the clone L cDNA. This primary translation product consists of an 185 amino acid polypeptide (19.8 kDa) including a 23 amino acid signal sequence. The presence of many serine and threonine residues may indicate an O-glycosylation. The predicted mature polypeptide shows an internal helical domain with 2 amphipathic alpha-helices. These might be involved in the association of Gra 2 with the membranous network within the parasitophorous vacuole.

Molecular structure of a Toxoplasma gondii dense granule antigen (GRA 5) associated with the parasitophorous vacuole membrane.

The P21 antigen of Toxoplasma gondii, defined by the monoclonal antibody TG17-113, has been described as a dense granule component, secreted in the parasitophorous vacuole during host cell invasion. The present work reports the cloning of the gene encoding the P21 antigen, for which we propose the name GRA 5. A cDNA library was screened with a rat antiserum raised against an HPLC fraction enriched in the P21 antigen. cDNA clones encoding GRA 5 were selected by antibody selection on the recombinant proteins. All these clones were incomplete at the 5' end. The 5' fragment of the longest cDNA clone isolated by this first screening was used as a probe in secondary screenings of cDNA and genomic DNA libraries. A genomic fragment containing the P21 gene and nearly full-length cDNAs have been isolated and sequenced. The gene encoding GRA 5 is 834 bp long and does not contain any intron. The deduced amino acid sequence of an open reading frame encoding 133 amino acids perfectly matched that of 5 peptides microsequenced from the native antigen. A N-terminal hydrophobic region was found to possess the characteristics of a signal peptide of 25 amino acids. A second hydrophobic domain, bordered by two hydrophilic regions strongly suggests a transmembrane region. This molecular structure is supported by ultrastructural studies showing the association of the P21 antigen with the parasitophorous vacuole membrane.

1,25-Dihydroxyvitamin D3 inhibits the expression of inducible nitric oxide synthase in rat central nervous system during experimental allergic encephalomyelitis.

The inducible form of nitric oxide synthase (iNOS) generates nitric oxide of which the excessive production is associated with central nervous system (CNS) inflammatory diseases. The investigation of iNOS expression during experimental allergic encephalomyelitis (EAE) of the Lewis rat demonstrated iNOS immunoreactivity and mRNA both during inflammatory bursts (days 12 and 23 post-immunization) and during the remission phase (day 18). iNOS expression was region-specific and expanded with time along a caudo-rostral axis, thus, correlating with the development of inflammatory infiltrates. Whereas cells of the monocyte/macrophage lineage continuously contributed to iNOS expression, astrocytes only expressed iNOS immunoreactivity or mRNA during the relapse (day 23). In order to investigate possible regulatory effects of 1,25-dihydroxyvitamin D3 (1,25-D3) on iNOS expression, rats were treated with the hormone after the beginning of clinical signs (days 11, 13, 19, 21 and 23 post-immunization), and areas of the CNS were examined at day 23. 1,25-D3 exerted a drastic inhibitory effect on iNOS expression, both at the protein and the mRNA levels. However, this effect was region-specific, and was most pronounced in the cerebellum and brainstem, but non-existent in cerebral cortex. iNOS down-regulation occurred in macrophages, activated microglia and astrocytes. The inhibition of iNOS expression in some CNS structures could account for the improvement of clinical signs observed in EAE-rats treated with 1,25-D3. Since 1,25-D3 can be synthesized by activated macrophages or microglia, our results support the hypothesis that this hormone might be implicated in the control of the CNS-specific immune responses. 1,25-D3 or its analogues could, thus, be of therapeutic value in the management of iNOS-associated diseases of the CNS.

A rapid percoll gradient procedure for the preparation of acetylcholine receptor-rich vesicles from Torpedo marmorata electric organ.

A rapid method for the isolation of acetylcholine receptor-rich membranes from Torpedo marmorata electric organ, using a Percoll density gradient, is presented. The preparation of purified membranes appeared on electron microscope examination as a homogeneous population of sealed vesicles, covered with the characteristic rosettes identified as acetylcholine receptor clusters. Biochemical characterization revealed an ?-bungarotoxin specific binding activity of 1.6-2.1 nmol/mg of protein, low acetylcholinesterase specific activity, a protein:lipid ratio (w/w) of 2.1 with high cholesterol content. Polyacrylamide gel electrophoresis under denaturing conditions showed the polypeptide bands characteristic of the receptor (?, ?, ? and ?), together with 43 kDa and ?100 kDa proteins (already described as ? and ?). The method is simple and rapid, and maintains constant osmotic conditions throughout. It thus represents an improvement over previous methods, and will be useful for routine preparation and specially for studying post-synaptic membrane components interactions.