Search

Abstract Background Lupus nephritis (LN) is an inflammatory disease of the kidneys affecting patients with systemic lupus erythematosus. Current immunosuppressive and cytotoxic therapies are associated with serious side effects and fail to protect 20–40% of LN patients from end-stage renal disease. In this study, we investigated whether a small heat shock protein, HSPB5, can reduce kidney inflammation and the clinical manifestations of the disease in NZB/W F1 mice. Furthermore, we investigated whether HSPB5 can enhance the effects of methylprednisolone, a standard-of-care drug in LN, in an endotoxemia mouse model. Methods NZB/W F1 mice were treated with HSPB5, methylprednisolone, or vehicle from 23 to 38 weeks of age. Disease progression was evaluated by weekly proteinuria scores. At the end of the study, the blood, urine, spleens, and kidneys were collected for the assessment of proteinuria, blood urea nitrogen, kidney histology, serum IL-6 and anti-dsDNA levels, immune cell populations, and their phenotypes, as well as the transcript levels of proinflammatory chemokine/cytokines in the kidneys. HSPB5 was also evaluated in combination with methylprednisolone in a lipopolysaccharide-induced endotoxemia mouse model; serum IL-6 levels were measured at 24 h post-endotoxemia induction. Results HSPB5 significantly reduced terminal proteinuria and BUN and substantially improved kidney pathology. Similar trends, although to a lower extent, were observed with methylprednisolone treatment. Serum IL-6 levels and kidney expression of BAFF, IL-6, IFNγ, MCP-1 (CCL2), and KIM-1 were reduced, whereas nephrin expression was significantly preserved compared to vehicle-treated mice. Lastly, splenic Tregs and Bregs were significantly induced with HSPB5 treatment. HSPB5 in combination with methylprednisolone also significantly reduced serum IL-6 levels in endotoxemia mice. Conclusions HSPB5 treatment reduces kidney inflammation and injury, providing therapeutic benefits in NZB/W F1 mice. Given that HSPB5 enhances the anti-inflammatory effects of methylprednisolone, there is a strong interest to develop HSBP5 as a therapeutic for the treatment of LN.

Lupus nephritis (LN) is a common and serious complication of systemic lupus erythematosus. The current treatments for LN are accompanied with severe immunotoxicity and have limits of effectiveness. Since our in vitro experiments demonstrated that a small heat shock protein (HSP), alpha-B crystallin (HSPB5; CRYAB), selectively modulates myeloid cells towards anti-inflammatory and tolerogenic phenotypes, the aim of this study was to investigate whether HSPB5 can attenuate the severity of LN. MRL/lpr mice were treated intravenously with HSPB5 at 2.5 or 10 μg/dose twice per week after disease onset, from 11 to 21 weeks of age. Disease progression was monitored by weekly measurements of proteinuria, and sera, spleens, and kidneys were collected for assessment at the terminal time point. Treatment with 10 μg HSPB5 substantially reduced endocapillary proliferation and tubular atrophy, which significantly reduced proteinuria and blood urea nitrogen (BUN). Compared to vehicle, 10 μg HSPB5 treatment substantially decreased activation/proliferation of splenocytes, increased IL-10+ macrophages, T and B regulatory cells (Treg, Breg), increased serum IL-10, and lowered expression of IL-6 in kidneys, which correlated with improved kidney function and pathology. This study demonstrated the utility of exogenous human HSPB5 to attenuate severe nephropathy in MRL/lpr mice and provides evidence in favour of a novel therapeutic approach for lupus nephritis.

Summarizes influenza activity in the United States from October 4, 1998 through February 27, 1999. Results of an investigation of an influenza outbreak at a long-term-care facility; Implication of the influenza outbreak; Vaccine effectiveness against influenza-like activities.

OBJECTIVES: This study determined infection risk for HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV) from needle reuse at a phlebotomy center that possibly exposed 3810 patients to infection. METHODS: We used a model for the risk of infection per blood draw, supplemented by subsequent testing results from 1699 patients. RESULTS: The highest risk of transmission was for HBV infection: 1.1 x 10(-6) in the best case and 1.2 x 10(-3) in the (unlikely) worst case. Subsequent testing yielded prevalence rates of 0.12%, 0.41%, and 0.88% for HIV, HBV, and HCV, respectively, lower than National Health and Nutrition Examination Survey III prevalence estimates. CONCLUSIONS: The infection risk was very low; few, if any, transmissions are likely to have occurred. [ABSTRACT FROM AUTHOR]

Vascular injury models are indispensable for studying thrombotic processes in vivo. Amongst the available methods for inducing thrombosis, laser-induced endothelial injury (LIEI) has several unique advantages. However, a lack of methodological standardization and expensive instrumentation remain significant problems decreasing reproducibility and impeding the adoption of LIEI in the wider scientific community. In this, study, we developed a standardized protocol for scanning laser-induced endothelial injury (scanning-LIEI) of murine mesenteric veins using the intrinsic 405 nm laser of a conventional laser scanning confocal microscope. We show that our model produces thrombi with prominent core-shell architectures and minimal radiation-related fluorescence artefacts. In comparison with previous methods, the scanning-LIEI model exhibits reduced experimental variability, enabling the demonstration of dose-response effects for anti-thrombotic drugs using small animal cohorts. Scanning-LIEI using the intrinsic 405 nm laser of a confocal laser scanning microscope represents a new method to induce standardized vascular injury with improved reproducibility of thrombus formation. The reduced need for instrument customisation and user experience means that this model could be more readily adopted in the research community., (© 2022. The Author(s).)

Competing Interests: The author declares no conflicts of interest.

In January 2020, Santa Clara County, California, USA, began identifying laboratory-confirmed coronavirus disease among residents. County staff conducted case and contact investigations focused on households and collected detailed case demographic, occupation, exposure, and outcome information. We describe the first 200 test-positive cases during January 31-March 20, 2020, to inform future case and contact investigations. Probable infection sources included community transmission (104 cases), known close contact with a confirmed case-patient (66 cases), and travel (30 cases). Disease patterns across race and ethnicity, occupational, and household factors suggested multiple infection risk factors. Disproportionately high percentages of case-patients from racial and ethnic subgroups worked outside the home (Hispanic [86%] and Filipino [100%]); household transmission was more common among persons from Vietnam (53%). Even with the few initial cases, detailed case and contact investigations of household contacts capturing occupational and disaggregated race and ethnicity data helped identify at-risk groups and focused solutions for disease control.

Context: In March, 2020, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19), was spreading in the Bay Area, especially in Santa Clara County, causing increases in cases, hospitalizations, and deaths., Program: The Association of Bay Area Health Officials (ABAHO) represents 13 Bay Area health jurisdictions., Implementation: On March 15, 2020, the local health officers of 7 ABAHO members (counties of Alameda, Contra Costa, Marin, San Francisco, San Mateo, and Santa Clara and the city of Berkeley) decided to issue legal orders on March 16 for 6.7 million residents to shelter in place to prevent the spread of SARS-CoV-2, the causal agent of COVID-19. The Bay Area was the first region in the United States to shelter in place, and within days, other regions in the United States followed., Evaluation: Subsequent comparative analyses have confirmed that acting early in issuing shelter-in-place orders prevented a large number of cases, hospitalizations, and deaths in the Bay Area throughout the United States. The quality of a decision-in this case, for crisis decision making-cannot be judged by the outcome. A good decision can have a bad outcome, and a bad decision can have a good outcome. The quality of a decision depends only on the quality of the decision-making process at the time the decision was made., Discussion: In this Field Report, we review how we made this collective decision. With the benefit of hindsight and reflection, we recount our story through the lens of public health legal authority, meta-leadership, and decision intelligence. Our purpose is to improve the crisis decision-making skills of public health officials by improving how we make high-stakes decisions each day in our continuing fight to contain the SARS-CoV-2 pandemic, to save lives, and to eliminate COVID-19 racial/ethnic inequities.

Objective: AIBP (apolipoprotein A-I binding protein) is an effective and selective regulator of lipid rafts modulating many metabolic pathways originating from the rafts, including inflammation. The mechanism of action was suggested to involve stimulation by AIBP of cholesterol efflux, depleting rafts of cholesterol, which is essential for lipid raft integrity. Here we describe a different mechanism contributing to the regulation of lipid rafts by AIBP. Approach and Results: We demonstrate that modulation of rafts by AIBP may not exclusively depend on the rate of cholesterol efflux or presence of the key regulator of the efflux, ABCA1 (ATP-binding cassette transporter A-I). AIBP interacted with phosphatidylinositol 3-phosphate, which was associated with increased abundance and activation of Cdc42 and rearrangement of the actin cytoskeleton. Cytoskeleton rearrangement was accompanied with reduction of the abundance of lipid rafts, without significant changes in the lipid composition of the rafts. The interaction of AIBP with phosphatidylinositol 3-phosphate was blocked by AIBP substrate, NADPH (nicotinamide adenine dinucleotide phosphate), and both NADPH and silencing of Cdc42 interfered with the ability of AIBP to regulate lipid rafts and cholesterol efflux., Conclusions: Our findings indicate that an underlying mechanism of regulation of lipid rafts by AIBP involves PIP-dependent rearrangement of the cytoskeleton.

Coronavirus disease 2019 (COVID-19), the respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in Wuhan, China and has since become pandemic. In response to the first cases identified in the United States, close contacts of confirmed COVID-19 cases were investigated to enable early identification and isolation of additional cases and to learn more about risk factors for transmission. Close contacts of nine early travel-related cases in the United States were identified and monitored daily for development of symptoms (active monitoring). Selected close contacts (including those with exposures categorized as higher risk) were targeted for collection of additional exposure information and respiratory samples. Respiratory samples were tested for SARS-CoV-2 by real-time reverse transcription polymerase chain reaction at the Centers for Disease Control and Prevention. Four hundred four close contacts were actively monitored in the jurisdictions that managed the travel-related cases. Three hundred thirty-eight of the 404 close contacts provided at least basic exposure information, of whom 159 close contacts had ≥1 set of respiratory samples collected and tested. Across all actively monitored close contacts, two additional symptomatic COVID-19 cases (i.e., secondary cases) were identified; both secondary cases were in spouses of travel-associated case patients. When considering only household members, all of whom had ≥1 respiratory sample tested for SARS-CoV-2, the secondary attack rate (i.e., the number of secondary cases as a proportion of total close contacts) was 13% (95% CI: 4-38%). The results from these contact tracing investigations suggest that household members, especially significant others, of COVID-19 cases are at highest risk of becoming infected. The importance of personal protective equipment for healthcare workers is also underlined. Isolation of persons with COVID-19, in combination with quarantine of exposed close contacts and practice of everyday preventive behaviors, is important to mitigate spread of COVID-19., Competing Interests: The authors have declared that no competing interests exist.

Background: Arterial thrombosis models are important for preclinical evaluation of antithrombotics but how anesthetic protocol can influence experimental results is not studied., Objectives: We studied how three most commonly used rodent anesthetics affect the induction of thrombosis and thrombus resolution with antiplatelet agent integrilin (Eptifibatide)., Methods: The Folts, electrolytic, and FeCl 3 models of carotid artery thrombosis were evaluated. The extent of blood flow reduction required to elicit cyclic flow reductions (CFR) was examined in the Folts model. The occlusion time and stability following electrolytic or FeCl 3 injury was assessed. The efficacy of Eptifibatide was studied in each cohort and clot composition following FeCl 3 application was assessed histologically., Results: Isoflurane and ketamine-xylazine (ket-x) elicited higher basal blood flow velocities. For reliable CFR in the Folts model, a higher degree of blood flow reduction was required under ket-x and isoflurane. For the FeCl 3 and electrolytic models, injury severity had to be increased in mice under ket-x anesthesia to achieve rapid occlusion. FeCl 3 -injured artery sections from ket-x and isoflurane-treated mice showed vessel dilatation and clots that were more fibrin/red-cell rich compared to pentobarbitone. Integrilin led to cycle abolishment for all three Folts-injury cohorts but for the electrolytic model a 2.5-fold higher dose was required to restore blood flow under pentobarbitone. Integrilin after FeCl 3 arterial injury was partially ineffective in isoflurane-treated mice., Conclusions: Anesthesia impacts rodent carotid artery occlusion experiments and alters integrilin efficacy. It is important to consider anesthetic protocols in animal experiments involving pharmacological agents for treatment of atherothrombosis.

Nanomedicine holds great promise for vascular disease diagnosis and specific therapy, yet rapid sequestration by the mononuclear phagocytic system limits the efficacy of particle-based agents. The use of low-fouling polymers, such as poly(ethylene glycol), efficiently reduces this immune recognition, but these nondegradable polymers can accumulate in the human body and may cause adverse effects after prolonged use. Thus, new particle formulations combining stealth, low immunogenicity and biocompatible features are required to enable clinical use. Here, a low-fouling particle platform is described using exclusively protein material. A recombinant protein with superior hydrophilic characteristics provided by the amino acid repeat proline, alanine, and serine (PAS) is designed and cross-linked into particles with lysine (K) and polyglutamic acid (E) using mesoporous silica templating. The obtained PASKE particles have low-fouling behavior, have a prolonged circulation time compared to albumin-based particles, and are rapidly degraded in the cell's lysosomal compartment. When labeled with near-infrared fluorescent molecules and functionalized with an anti-glycoprotein IIb/IIIa single-chain antibody targeting activated platelets, the particles show potential as a noninvasive molecular imaging tool in a mouse model of carotid artery thrombosis. The PASKE particles constitute a promising biodegradable and versatile platform for molecular imaging of vascular diseases.

Objective: To investigate Acinetobacter baumannii infection, colonization, and transmission related to a long-term care facility (LTCF) providing subacute care (facility A)., Methods: We reviewed facility A and affiliated local hospital records for facility A residents with A. baumannii isolated during the period January 2009 through February 2010 and compared A. baumannii antimicrobial resistance patterns of residents with those of hospital patients. During March 2010, we implemented a colonization survey of facility A residents who received respiratory support or who could provide sputum samples and looked for A. baumannii colonization risks. Available clinical and survey isolates underwent pulsed-field gel electrophoresis (PFGE); PFGE strains were linked with overlapping stays to identify possible transmission., Results: During the period January 2009 through February 2010, 33 facility A residents had A. baumannii isolates; all strains were multidrug resistant (MDR), which was a significantly higher prevalence of MDR strains than that found among isolates from hospital patients (81 [66%] of 122 hospital patient isolates were MDR; P < .001). The sputum survey found that 14 (20%) of 70 residents had A. baumannii colonization, which was associated with ventilator use (adjusted odds ratio, 4.24 [95% confidence interval, 1.06-16.93]); 12 (86%) of 14 isolates were MDR. Four facility A resident groups clustered with 3 PFGE strains and overlapping stays. One of these facility A residents also clustered with 3 patients at an affiliated hospital., Conclusions: We documented substantial MDR A. baumannii infections and colonization with probable intra- and interfacility spread associated with a single LTCF providing subacute care. Given the limited infection prevention and antimicrobial stewardship resources in such settings, regional collaborations among facilities across the spectrum of health care are needed to address this MDR threat.

Cellular injury causes a myriad of processes that affect proteostasis. We describe nucleocytoplasmic coagulation (NCC), an intracellular disulfide-dependent protein crosslinking event occurring upon late-stage cell death that orchestrates the proteolytic removal of misfolded proteins. In vitro and in vivo models of neuronal injury show that NCC involves conversion of soluble intracellular proteins, including tubulin, into insoluble oligomers. These oligomers, also seen in human brain tissue following neurotrauma, act as a cofactor and substrate for the plasminogen-activating system. In plasminogen(-/-) mice, levels of misfolded β-tubulin were elevated and its clearance delayed following neurotrauma, demonstrating a requirement for plasminogen in the removal of NCC constituents. While additional in vivo studies will further dissect this phenomenon, our study clearly shows that NCC, a process analogous to the formation of thrombi, generates an aggregated protein scaffold that limits release of cellular components and recruits clearance mechanisms to the site of injury., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Current optical methods to collect Nomarski differential interference contrast (DIC) or phase images with a transmitted light detector (TLD) in conjunction with confocal laser scanning microscopy (CLSM) can be technically challenging and inefficient. We describe for the first time a simple method that combines the use of the commercial product QPm (Iatia, Melbourne Australia) with brightfield images collected with the TLD of a CLSM, generating DIC, phase, Zernike phase, dark-field or Hoffman modulation contrast images. The brightfield images may be collected at the same time as the confocal images. This method also allows the calculation of contrast-enhanced images from archival data. The technique described here allows for the creation of contrast-enhanced images such as DIC or phase, without compromising the intensity or quality of confocal images collected simultaneously. Provided the confocal microscope is equipped with a motorized z-drive and a TLD, no hardware or optical modifications are required. The contrast-enhanced images are calculated with software using the quantitative phase-amplitude microscopy technique (Barone-Nugent et al., 2002). This technique, being far simpler during image collection, allows the microscopist to concentrate on their confocal imaging and experimental procedures. Unlike conventional DIC, this technique may be used to calculate DIC images when cells are imaged through plastic, and without the use of expensive strain-free objective lenses.

Fluorescence resonance energy transfer (FRET) from a donor-labelled molecule to an acceptor-labelled molecule is a useful, proximity-based fluorescence tool to discriminate molecular states on the surface and in the interior of cells. Most microscope-based determinations of FRET yield only a single value, the interpretation of which is necessarily model-dependent. In this paper we demonstrate two new measurements of FRET heterogeneity using selective donor photobleaching in combination with synchronous donor/acceptor detection based on either (1) full kinetic analysis of donor-detected and acceptor-detected donor photobleaching or (2) a simple time-based ratiometric approach. We apply the new methods to study the cell surface distribution of concanavalin A yielding estimates of FRET and non-FRET population distributions, as well as FRET efficiencies within the FRET populations.

The Netrin receptor Deleted in colon cancer (Dcc) has been shown to play a pivotal role in the guidance of nascent axons towards the ventral midline in the developing nervous systems of both vertebrates and invertebrates. In contrast, the function during embryogenesis of a second Dcc-like Netrin receptor Neogenin has not yet been defined. We used antisense morpholino oligonucleotides to knockdown Neogenin activity in zebrafish embryos and demonstrate that Neogenin plays an important role in neural tube formation and somitogenesis. In Neogenin knockdown embryos, cavitation within the neural rod failed to occur, producing a neural tube lacking a lumen. Somite formation was also defective, implicating Neogenin in the migration events underlying convergent extension during gastrulation. These observations suggest a role for Neogenin in determining cell polarity or migrational directionality of both neuroectodermal and mesodermal cells during early embryonic development.

The spi1 (pu.1) gene has recently been identified as a useful marker of early myeloid cells in zebrafish. To enhance the versatility of this organism as a model for studying myeloid development, the promoter of this gene has been isolated and characterized. Transient transgenesis revealed that a 5.3 kilobase promoter fragment immediately upstream of the spi1 coding sequence was sufficient to drive expression of enhanced green fluorescent protein (EGFP) in injected embryos in a manner that largely recapitulated the native spi1 gene expression pattern. This fragment was successfully used to produce a germ line transgenic line of zebrafish with EGFP-expressing myeloid cells. These TG(spi1:EGFP)pA301 transgenic zebrafish represent a valuable tool for further studies of myeloid development and its perturbation.

Background: An anesthesiologist was diagnosed as having acute hepatitis C 3 days after providing anesthesia during the thoracotomy of a 64-year-old man (patient A). Eight weeks later, patient A was diagnosed as having acute hepatitis C., Methods: We performed tests for antibody to hepatitis C virus (HCV) on serum samples from the thoracotomy surgical team and from surgical patients at the 2 hospitals where the anesthesiologist worked before and after his illness. We determined the genetic relatedness of the HCV isolates by sequencing the quasispecies from hypervariable region 1., Results: Of the surgical team members, only the anesthesiologist was positive for antibody to HCV. Of the 348 surgical patients treated by him and tested, 6 were positive for antibody to HCV. Of these 6 patients, isolates from 2 (patients A and B) were the same genotype (1a) as that of the anesthesiologist. The quasispecies sequences of these 3 isolates clustered with nucleotide identity of 97.8% to 100.0%. Patient B was positive for antibody to HCV before her surgery 9 weeks before the anesthesiologist's illness onset. The anesthesiologist did not perform any exposure-prone invasive procedures, and no breaks in technique or incidents were reported. He denied risk factors for HCV., Conclusions: Our investigation suggests that the anesthesiologist acquired HCV infection from patient B and transmitted HCV to patient A. No further transmission was identified. Although we did not establish how transmission occurred in this instance, the one previous report of bloodborne pathogen transmission to patients from an anesthesiologist involved reuse of needles for self-injection.

A questionnaire regarding tolerability and adherence was administered for 5 days to hospital employees who received azithromycin prophylaxis during a hospitalwide outbreak of a pertussis-like illness. Analysis of the 239 responses from those having received prophylactic azithromycin determined that it was well tolerated and accounted for a minimal loss of days worked; 81.5% were fully adherent with the regimen.

The chimeric monoclonal antibody KM871, directed against the G(D3) antigen, is under evaluation for its potential to target melanoma. To facilitate the in vivo evaluation of biodistribution properties and measurement of pharmacokinetics, KM871 was radiolabeled with (125)I via tyrosine residues and with (111)In via the bifunctional metal ion chelator C-functionalized trans-cyclohexyl diethylenetriaminepentaacetic acid (CHX-A"-DTPA) to lysine residues. Using antigen-positive SK-MEL-28 melanoma cells, immunoreactivities of 42 and 40% cell binding were obtained, respectively, for the two radioconjugates. Binding was enhanced in the presence of added unlabeled antibody. A humanized A33 antibody was similarly labeled with the two isotopes and used as a control. To determine and compare in vivo biodistribution characteristics of KM871 radiolabeled with (111)In or (125)I, mixtures of the radioconjugates were injected i.v. into BALB/c nude mice bearing G(D3)-positive-SK-MEL-28 melanoma xenografts. Gamma camera images were acquired; groups of five mice were sacrificed at various time intervals, and tumors, blood, and tissues were analyzed. (111)In-labeled CHX-A"-DTPA-KM871 showed a maximum tumor uptake of 41.9 +/- 7.0% injected dose/g at 72 h with prolonged retention over a 15-day period. The tumor:blood ratio was 3:1 by 72 h, and higher ratios were observed at later time points. No abnormal accumulation of (111)In-labeled conjugate was found in normal tissues. In contrast, there was little accumulation of (125)I-labeled KM871 in the same tumors. The specificity of antibody localization was confirmed by the low tumor uptake values for radiolabeled control antibody. Gamma camera imaging demonstrated excellent uptake of (111)In-labeled CHX-A"-DTPA-KM871 in the xenografts. Chromatographic analyses of xenograft cytosolic extracts demonstrated tumor internalization and catabolism of radiolabeled KM871 with the formation of small molecular weight metabolites. Laser scanning confocal microscopy demonstrated that the majority of intracellular KM871 is localized to lysosomes. Despite the catabolism of the radioconjugate, a dose-dependent increase in KM871 tumor localization was shown through immunohistochemical examination of xenograft biopsies. This study demonstrates for the first time the in vivo localization of a radiolabeled anti-G(D3) monoclonal antibody to G(D3)-expressing xenografts using gamma camera scanning techniques and tumor cell internalization of KM871 tagged with a green fluorescent dye, Alexa Fluor 488, through confocal microscopy. KM871 has potential for targeting tumors in patients with melanoma.

The murine A33 antigen is emerging as a definitive marker of intestinal epithelial cells. Cloning and sequence determination of cDNAs encoding mA33 antigen predict a novel type 1 transmembrane protein of 298 amino acids, comprising an extracellular domain with two immunoglobulin-like domains, a single-span transmembrane domain, and a highly acidic cytoplasmic domain. On the basis of conservation of amino acid sequence and genomic structure, the mA33 antigen is a member of a growing subfamily within the immunoglobulin superfamily, which includes transmembrane proteins CTX/ChT1, CTM/CTH, and CAR. During embryonic development, mA33 antigen expression is first observed in the inner cell mass of blastocysts before implantation. Intestinal expression of mA33 antigen is initiated in the hindgut at E14.5 and increases steadily throughout late embryonic and postnatal life into adulthood. The protein is specifically expressed on the basolateral surfaces of intestinal epithelial cells of all lineages, independent of their position along the rostrocaudal and crypt-villus axes. Thus the mA33 antigen appears to be a novel marker for both proliferating and differentiating intestinal epithelial cells.

Context: Salmonella serotype Typhimurium definitive type 104 (DT104), with resistance to 5 drugs (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline), has emerged as the most common multidrug-resistant Salmonella strain in the United States. However, illnesses resulting from this strain have not been associated definitively with a source in this country., Objective: To determine the source of 2 outbreaks of Salmonella Typhimurium DT104., Design: Matched case-control study conducted between March 24 and April 5, 1997 (outbreak 1), enhanced surveillance for new cases dating from February 1, 1997 (outbreak 2), and environmental and laboratory investigations., Setting and Participants: The case-control study included residents of 2 adjacent counties in northern California infected with the outbreak strain of Salmonella Typhimurium var Copenhagen and age-matched controls. For enhanced surveillance, a case was defined as Salmonella Typhimurium infection in a person exposed to fresh Mexican-style cheese., Main Outcome Measures: Risk factors for infection and source of implicated food., Results: Outbreak 1 peaked in February 1997; 31 patients were confirmed by culture as having Salmonella Typhimurium var Copenhagen infection, isolates of which showed indistinguishable pulsed-field gel electrophoresis (PFGE) patterns. The outbreak strain was phage type DT104 with the 5-drug resistance pattern. Sixteen cases and 25 controls were enrolled in the case-control study; 15 of 16 Salmonella Typhimurium var Copenhagen cases compared with 14 of 24 matched controls reported eating unpasteurized Mexican-style cheese, (matched odds ratio, 7.9; 95% confidence interval, 1.1-354.9). Enhanced surveillance uncovered outbreak 2, which peaked in April 1997 and was caused by a non-Copenhagen variant of Salmonella Typhimurium. During outbreak 2, Salmonella Typhimurium was isolated from 79 persons who ate fresh Mexican-style cheese from street vendors and from cheese samples and raw milk. The PFGE pattern of the milk isolate matched 1 of the 3 patterns recovered from patients; all strains were phage type DT104b with the 5-drug resistance pattern., Conclusion: Raw-milk products pose a risk for multidrug-resistant Salmonella Typhimurium DT104 infections.

Background: Escherichia coli O157:H7 infections have traditionally been associated with animal products, but outbreaks associated with produce have been reported with increasing frequency. In fall 1996, a small cluster of E. coli O157:H7 infections was epidemiologically linked to a particular brand (brand A) of unpasteurized apple juice., Objective: To define the extent of the outbreak, confirm the source, and determine how the apple juice became contaminated., Design: Descriptive epidemiologic study and traceback investigation., Setting: Western United States and British Columbia, Canada., Patients: Patients with E. coli O157:H7 infection who were exposed to brand A apple juice., Measurements: Clinical outcome and juice exposure histories of case-patients, pulsed-field gel electrophoresis of case and juice isolates, and juice production practices., Results: Seventy persons with E. coli O157:H7 infection and exposure to brand A unpasteurized apple juice were identified. Of these persons, 25 (36%) were hospitalized, 14 (20%) developed the hemolytic uremic syndrome, and 1 (1%) died. Recalled apple juice that was produced on 7 October 1996 grew E. coli O157:H7 with a pulsed-field gel electrophoresis pattern indistinguishable from that of case isolates. Apple juice produced on 7 October 1996 accounted for almost all of the cases, and the source of contamination was suspected to be incoming apples. Three lots of apples could explain contamination of the juice: Two lots originated from an orchard frequented by deer that were subsequently shown to carry E. coli O157:H7, and one lot contained decayed apples that had been waxed., Conclusions: Standard procedures at a state-of-the-art plant that produced unpasteurized juices were inadequate to eliminate contamination with E. coli O157:H7. This outbreak demonstrated that unpasteurized juices must be considered a potentially hazardous food and led to widespread changes in the fresh juice industry.

Endothelin-1 (ET-1) has been shown to potentiate cholinergic neurotransmission in human bronchus as well as in airways from a variety of animal species, suggesting that ET receptors exist prejunctionally on airway cholinergic nerves. We have successfully isolated and maintained rat tracheal para-sympathetic neurons in culture. Most cultured cells were associated with specific fluorescence for the nerve cell marker protein gene product 9.5 (PGP 9.5). These cultures contained a high proportion of parasympathetic neurons. Importantly, specific immunofluorescent antibodies for ETB receptors were colocalized with those for PGP 9.5. Therefore, for the first time, ETB receptors have been shown to exist on airway parasympathetic neurons in culture.

Osteoclasts are multinuclear bone-resorbing cells which contain abundant mitochondria. Morphological studies have suggested that a correlation may exist between mitochondrial concentration and bone resorption by osteoclasts. However, investigation of mitochondrial transmembrane potential (delta psi) and volume has been hampered by the difficulty in obtaining a sufficient number of osteoclasts for assessing these characteristics by flow cytometric analysis. In this study, we have used confocal laser scanning microscopy after loading the cells with Rhodamine 123 and 10-nonyl Acridine Orange to record mitochondrial delta psi and volume, respectively, in isolated rat osteoclasts cultured on bovine bone slices. Optimal staining conditions were found to be 10 micrograms ml-1 for 40 min for Rhodamine, and 1 microM for 10 min for the 10-nonyl Acridine Orange derivative. Two osteoclast populations, whose shape seemed to reflect bone resorption and migratory functions, were identified depending on their shape and on the distribution of the two dye probes. 'Round-shaped' osteoclasts had significantly higher mitochondrial delta psi and volume in the apical regions than in the basolateral portions (p < 0.00001). In contrast, mitochondrial delta psi and volume in 'irregular-shaped' osteoclasts were rather evenly distributed in both these regions (p > 0.05). Our results indicate that there is an apical polarization of mitochondria in osteoclasts corresponding to the energy demands associated with bone resorption.

Conventional ways of demonstrating and analysing the components of osseous tissue have always been hampered by the difficulty of physically sectioning bone. In this study, we have used Acridine Orange staining of 100-micron-thick unembedded bone slices and then assessed the cellular and tissue architecture by confocal microscopy. The result showed the Acridine Orange, by differential staining of the cellular nucleic acids, permits ready assessment of cell shape and cell organization as well as variations in growth patterns. Our studies have provided a new and relatively easy way of assessing the morphology of bone specimens by rendering unnecessary the need for embedding, decalcification and thin sectioning of the osseous tissue.

We describe the first direct visualization of Ca2+ oscillations in the perfused whole rat heart. Dye loading at a low temperature and enhanced optical-section techniques of confocal microscopy by elimination of the refractive index mismatch with use of saline-immersible objective lens enabled us to image multiple Ca2+ waves in the subepicardial myocardium of the fluo 3-loaded heart. These Ca2+ waves were sporadically seen even with a physiological extracellular Ca2+ perfusion in either a paced or an arrested heart and propagated beyond cellular boundaries within the three-dimensional structures of cardiac muscle. Under these conditions, the velocity of wave propagation was 60-100 microns/s and the frequency of initiation was relatively low (< 2 Hz). With an increase in extracellular Ca2+ concentration, however, the waves became more prevalent and tended to be multifocal, and an increasing fraction of the waves exhibited faster propagation velocities and higher frequencies. These results suggest that perfused rat hearts exhibit spontaneous Ca2+ waves in an apparently resting state and that under Ca(2+)-overload conditions the multifocal and high-frequency waves become more widespread in the heart syncytium, which may provide an understanding of the ionic basis for the summation of afterdepolarizations and triggering of arrhythmias seen under pathological conditions.

Laser scanning confocal microscopy of the Ca(2+)-sensitive fluorophore fluo-3 has been used to investigate spontaneous and propagated calcium release at high temporal and spatial resolution in enzymatically dispersed rat cardiomyocytes. Waves of fluorescence which propagated throughout the cytosol were evident in spontaneously contracting cardiac cells containing fluo-3, but not in cells containing Ca(2+)-insensitive fluorophores [2',7'-bis (carboxyethyl)-5,6-carboxyfluorescein, SNARF-1, rhodamine-123, or tetramethylrhodamine-labeled dextran]. These waves represent localized areas of elevated [Ca2+] [975 +/- 13 (SE) nM, range 800-1,500 nM; n = 16 cells]. Ca2+ waves were initiated by the spontaneous release of Ca2+ from the sarcoplasmic reticulum (SR) and propagated through cells at rates of 50-150 microns/s. Ca2+ waves were usually initiated at the cell ends, but multiple and variable initiation foci were observed in some cells. Where waves intersected within a single cell there was extinction of wave propagation, confirming the SR as the direct source of Ca2+ and revealing a refractory period in SR Ca2+ release. In some cells high-frequency Ca2+ waves lead to synchronized elevation of [Ca2+] throughout the entire cytosol and within the time period associated with cell depolarization. These observations support the hypothesis that some cardiac arrhythmias are initiated by spontaneous and propagated Ca2+ release and involve subsequent depolarization, global elevation of intracellular [Ca2+], and cell contraction.

Phototropism and gravitropism in the shoots and roots of higher plants are the result of asymmetric growth. This is explained by the redistribution of growth regulators following exposure to gravity or unilateral light (the Cholodny-Went hypothesis). The positive phototropism and the negative geotropism of grass seedling coleoptiles are believed to result from lateral movement of auxin from the irradiated to the shaded side and from the upper to the lower side, respectively. Many physiological processes in plants, including auxin-induced cell elongation, are reported to be under the control of calcium. Added auxin triggers oscillations in cytosolic free calcium ([Ca2+]cyt) and cytosolic pH (pHcyt) in epidermal cells of maize coleoptiles. Until recently, it has not been possible to visualize these changes spatially with the commonly used fluorescent cation indicators. Using a scanning laser confocal microscope, a new visible wavelength Ca2+ probe fluo-3 and the fluorescent pH indicator BCECF, we have recorded rapid light-induced increases in [Ca2+]cyt and a lowering of pHcyt of cells on the shaded side of maize coleoptiles. In horizontally orientated coleoptiles, [Ca2+]cyt increases and pHcyt decreases in the more rapidly elongating cells on the lower side. For the first time, rapid changes in [Ca2+]cyt and pHcyt are correlated directly with increases in cell elongation stimulated by light and gravity.

Laser-scanning confocal microscopy has been used in conjunction with Fluo-3, a highly fluorescent visible wavelength probe for Ca2+, to visualize Ca2(+)-dynamics in the function of living plant cells. This combination has overcome many of the problems that have limited the use of fluorescence imaging techniques in the study of the role of cations (Ca2+ and H+) in plant cell physiology and enables these processes to be studied in single cells within intact plant tissue preparations. Maize coleoptiles respond to application of ionophores and plant growth hormones with elevations in cytosolic Ca2+ that can be resolved with a high degree of spatial resolution and can be interpreted quantitatively.