Biochemical analysis and decomposition products of indocyanine green in relation to solvents, dye concentrations and laser exposure.

PURPOSE: To investigate pH, ions, osmolarity and precipitation of indocyanine green (ICG), as well as the profile of ICG decomposition products (DPs) after laser exposure and the interaction with quenchers. METHODS: ICG was diluted in water, 5% glucose (GL) or balanced salt solution (BSS) to achieve concentrations of 2.5, 1, 0.25 and 0.1 mg/ml. Osmolarity, pH and precipitation were analyzed immediately and after 24 h. Precipitation analyses were done with a scanning electron microscope. Anion and iodate analyses of ICG and infracyanine green (IfCG) were performed by capillary zone electrophoresis. With regard to DPs, 0.5 mg/ml of ICG was assessed with high-performance liquid chromatography (HPLC) after 810-nm laser irradiation. DP profiles were evaluated with ICG dilution in quenchers (Trolox, histidine and DABCO) in 3 concentrations (0.1, 1 and 10 M). RESULTS: BSS promoted iso-osmotic ICG solutions of 208 mOsm (147-266) compared to GL with 177 mOsm. BSS solutions had a higher physiological pH of 7.2 compared with the GL one of 6.55. ICG precipitated more when diluted with BSS (5.95 mg); in contrast, GL showed less precipitate (3.6 mg). IfCG has no iodine derivates and other ICGs have an average 4.6% of iodate derivates. From HPLC analysis, 5 DPs were observed. The rate of DPs was higher when BSS was used (p < 0.05). Five DPs have been generated with ICG, and they may be altered with the quenchers DABCO, histidine and Trolox. CONCLUSIONS: BSS dilution induces more precipitation and DPs. ICG dilution in any solvent induces DPs. Quencher use reduces the amount of toxic DPs.

Changes in cell wall synthesis and ultrastructure during paradoxical growth effect of caspofungin on four different Candida species.

Paradoxical growth (PG) has been described for echinocandins and is characterized by cell growth at drug concentrations above the MIC. In this study, two isolates each of Candida albicans, C. tropicalis, C. orthopsilosis, and C. parapsilosis, all of which displaying PG in response to caspofungin, were subjected to MIC, minimal fungicidal concentration (MFC), and time-kill curve assays to evaluate the levels of PG. Cell wall components and ultrastructural modifications of the PG cells were also investigated. The results showed that when cell growth and survival were evaluated by MFC or time-kill curve assays, high concentrations of caspofungin did not show fungicidal activity against PG cells. Furthermore, for C. parapsilosis and C. orthopsilosis, time-kill curves were more discriminatory than MFCs in detecting the PG effect. The four different Candida species studied demonstrated similar alterations in cell wall components and ultrastructure associated with PG. In PG cells, ß-1,3-glucan content decreased from 2.7- to 7.8-fold, whereas chitin content increased from 4.0- to 6.6-fold. An electron microscopy study of the PG cells revealed morphological alterations, clumping of cells, enlarged cells, the absence of filamentation, abnormal septa, and accumulation of chitin in the cell wall. Also, PG cells basically exhibited a single dark high-density layer in the cell wall, indicating the loss of the ß-1,3-glucan layer. Our results present novel details about the ultrastructural alterations that occur in C. albicans, C. parapsilosis, C. orthopsilosis, and C. tropicalis during PG and show that chitin is the major component of the cell walls of PG cells. Stimulation of chitin synthesis may represent a rescue mechanism against caspofungin activity.

Biofilm production and evaluation of antifungal susceptibility amongst clinical Candida spp. isolates, including strains of the Candida parapsilosis complex.

Candida cells can form biofilms that frequently are sources of infections and are less susceptible to antifungal drugs. Some authors have reported that Candida orthopsilosis and Candida metapsilosis isolates are not able to produce biofilms in vitro and there are no studies available on biofilm susceptibility for these species to antifungals. The aims of this study were to (i) quantify Candida spp. biofilms in vitro, and (ii) test the in vitro susceptibilities of Candida spp. biofilms to fluconazole (FLC) and amphotericin B (AMB). Isolates studied included four Candida albicans, six C. tropicalis, seven C. parapsilosis, eight C. orthopsilosis, and five C. metapsilosis. We compared two different methods to evaluate biofilm production, i.e., crystal violet (CV) staining and XTT-reduction assays (XTT). Scanning electron microscopy (SEM) was used to observe high, medium and low biofilm producing isolates screened by these two methods. To determine the minimum biofilm eradication concentration (MBEC) for FLC and AMB, XTT-reduction assay was used to measure cell metabolic activity. Biofilm quantification by CV and XTT showed that C. tropicalis isolates were the highest biofilm producer, followed by C. albicans, C. parapsilosis, C. orthopsilosis and C. metapsilosis. Examination of SEM images revealed that the extent of biofilms formed by high, medium, and low producers was highly correlated to the results generated by CV assay. Biofilm of all the isolates evaluated were resistant to FLC (MBEC(80) ≥ 256 ug/ml) but, in general, susceptible to AMB, except for six C. parapsilosis strains (MBEC(80) ≥ 8 ug/ml).

Experimental investigation of needles, syringes and techniques for intravitreal injections.

BACKGROUND: To assess the techniques and materials used in intravitreal injections. DESIGN: Descriptive study realized at the Vision Institute of the Federal University of São Paulo, Brazil. SAMPLES: Different brands of needles and syringes, as well as enucleated porcine eyeballs. METHODS: The ultra-structures of commonly used needles were analysed by scanning electron microscope, and they were compared using different criteria, such as irregularities and debris from the lubrication process. The scleral incision was also assessed using needles of different brands and sizes. Accuracies in drug administration were studied by comparing the residual and delivered volume of needles and also by the analysis of reflux after intravitreal injections. MAIN OUTCOME MEASURES: Efficiency and quality of materials used in intravitreal injections. RESULTS: Ultra-structure analyses showed that all needles had different types of irregularities. Some photographs showed debris from the lubrication process, especially in BD needles. Scleral incision analysis showed a tendency of reducing the ocular damage with increasing gauge (P=0.024). The investigation of delivery accuracy showed that almost all needles underdosed the amount injected (P<0.05), and that the reflux could be minimized by tunnelled injections with thinner needles. CONCLUSION: Needles used in intravitreal injections possess many irregularities in their structures, which may cause different injection outcomes. Analyses of scleral incisions showed that the larger the needle gauge, the lesser the scleral damage and the risk of complications. Moreover, drug administration inaccuracies might be one of the causes for some unsuccessful attempts of treatment.

Fibrinolytic activity is associated with presence of cystic medial degeneration in aneurysms of the ascending aorta.

AIMS: Thoracic ascending aortic aneurysms (TAA) are characterized by elastic fibre breakdown and cystic medial degeneration within the aortic media, associated with progressive smooth muscle cell (SMC) rarefaction. The transforming growth factor (TGF)-ß/Smad2 signalling pathway is involved in this process. Because the pericellular fibrinolytic system activation is able to degrade adhesive proteins, activate matrix metalloproteinase (MMP), induce SMC disappearance and increase the bioavailability of TGF-ß, the aim was to investigate the plasminergic system in TAA. METHODS AND RESULTS: Ascending aortas [21 controls and 19 TAAs (of three different aetiologies)] were analysed. Immunohistochemistry showed accumulation of t-PA, u-PA and plasmin in TAAs, associated with residual SMCs. Overexpression of t-PA and u-PA was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting and zymography on TAA extracts and culture medium conditioned by TAA. Plasminogen was present on the SMC surface and inside cytoplasmic vesicles, but plasminogen mRNA was undetectable in the TAA medial layer. Plasmin-antiplasmin complexes were detected in TAA-conditioned medium and activation of the fibrinolytic system was associated with increased fibronectin turnover. Fibronectin-related material was detected immunohistochemically in dense clumps around SMCs and colocalized with latent TGF-ß binding protein-1. CONCLUSIONS: The fibrinolytic pathway could play a critical role in TAA progression, via direct or indirect impact on ECM and consecutive modulation of TGF-ß bioavailability.

Preclinical investigation of the retinal biocompatibility of six novel vital dyes for chromovitrectomy.

PURPOSE: To investigate the retinal biocompatibility of six novel vital dyes for chromovitrectomy. METHODS: An amount of 0.05 mL of 0.5% and 0.05% light green (LG), fast green (FG), Evans blue (EB), brilliant blue (BriB), bromophenol blue (BroB), or indigo carmine (IC) was injected intravitreally in the right eye, whereas in the left eye balanced salt solution was applied for control in rabbits' eyes. Clinical examination, fluorescein angiography, histology with light microscopy, and transmission electron microscopy were performed after 1 and 7 days. Retinal cell layers were evaluated for morphologic alterations and number of cells. The electroretinographic changes were assessed at baseline, 24 hours and 7 days. RESULTS: Fluorescein angiography disclosed hypofluorescent spots only in the 0.5% EB group. Light microscopy and transmission electron microscopy disclosed slight focal morphologic changes in eyes exposed to 0.05% IC, FG, BriB, similar to the control at 1 and 7 days. In the lower dose groups, EB, LG, and BroB caused substantial retinal alterations by light microscopy. At the higher dose, BroB and EB produced diffuse cellular edema and vacuolization within the ganglion cells, bipolar cells, and photoreceptors. FG and IC at 0.5% caused slight retinal alterations similar to balanced salt solution injection. LG at 0.5% caused diffuse vacuolization of bipolar cells after 1 and 7 days. Injection of 0.5% EB caused a significant decrease in neuroretinal cell counts in comparison to control eyes in the 7-day examination (P < 0.05). Electroretinography revealed intermittent prolonged latency and decreased amplitude in eyes injected with 0.5% EB, LG, BriB, and BroB, while at the lower dose, only LG and EB induced few functional changes. CONCLUSION: The progressive order of retinal biocompatibility, from safest to most toxic, was IC, FG, BriB, BroB, LG, EB.

B-1 lymphocytes increase metastatic behavior of melanoma cells through the extracellular signal-regulated kinase pathway.

Increasing evidence indicates that tumors require a constant influx of myelomonocytic cells to support their malignant behavior. This is caused by tumor-derived factors, which recruit and induce functional differentiation of myelomonocytic cells, most of which are macrophages. Although myeloid lineages are the classical precursors of macrophages, B-lymphoid lineages such as B-1 cells, a subset of B-lymphocytes found predominantly in pleural and peritoneal cavities, are also able to migrate to inflammatory sites and differentiate into mononuclear phagocytes exhibiting macrophage-like phenotypes. Here we examined the interplay of B-1 cells and tumor cells, and checked whether this interaction provides signals to influence melanoma cells metastases. Using in vitro coculture experiments we showed that B16, a murine melanoma cell line, and B-1 cells physically interact. Moreover, interaction of B16 with B-1 cells leads to up-regulation of metastasis-related gene expression (MMP-9 and CXCR-4), increasing its metastatic potential, as revealed by experimental metastases assays in vivo. We also provide evidence that B16 cells exhibit markedly up-regulated phosphorylation of the extracellular signal-regulated kinase (ERK) when cocultured with B-1 cells. Inhibition of ERK phosphorylation induced by B-1 cells with inhibitors of MEK1/2 strongly suppressed the induction of MMP-9 and CXCR-4 mRNA expression and impaired the increased metastatic behavior of B16. In addition, constitutive levels of ERK1/2 phosphorylation in B-1 cells are necessary for their commitment to affect the metastatic potential of B16 cells. Our findings show for the first time that B-1 lymphocytes can contribute to tumor cell properties required for invasiveness during metastatic spread.

Escherichia coli strains of serotype O51:H40 comprise typical and atypical enteropathogenic E. coli strains and are potentially diarrheagenic.

Escherichia coli strains of serotype O51:H40 were studied with regard to the presence of several virulence properties and their genetic diversity and enteropathogenicity in rabbit ileal loops. This serotype encompasses potential enteropathogenic strains mostly classified as being atypical enteropathogenic E. coli (EPEC) strains, which are genetically closer to enterohemorrhagic E. coli than to typical EPEC strains.

Damaging effects of intense repetitive treadmill running on murine intestinal musculature.

Several gastrointestinal symptoms associated with prolonged intense exercise (IE) have been reported, although the mechanisms underlying its effects on the intestine remain poorly understood. The aim of the present study was to investigate whether IE may induce oxidative stress in the intestine, as well as its possible relationship with intestinal signaling impairments, leading to contractile disturbances. C57BL/6 mice were submitted to 4 days (EX.4D) and 10 days (EX.10D) of IE. The daily exercise session consisted of a running session until exhaustion, with the treadmill speed set at 85% of each animal's maximum velocity. The decrease in exhaustion time was exponential, and the reduction in the maximum velocity, as assessed by an incremental test, was higher in EX.4D than in EX.10D animals. The ileum mucosa layer was partially destroyed after 4 days of IE, where 37% and 11% muscle layer atrophies were observed in EX.4D and EX.10D animals, respectively. Ileum contractility was significantly impaired in the EX.4D animal group, with reduced efficacy for carbachol, bradykinin, and KCl signaling associated with a decrease in lipid peroxidation and with no alteration of protein oxidation. Intestinal myocytes from EX.10D animals displayed areas containing structurally disorganized mitochondria, which were associated with increased levels of protein oxidation, without alteration of contractility, except for a reduction in the potency of bradykinin signaling. Finally, no clear relationship between ileum contractility and oxidative stress was shown. Together, these results argue in favor of significant functional, biochemical, and morphological disturbances caused by exercise, thus demonstrating that intestinal tissue is very sensitive to exercise.

Gamma-radiation induces apoptosis via sarcoplasmatic reticulum in guinea pig ileum smooth muscle cells.

We investigated the effects of gamma-radiation on cells isolated from the longitudinal smooth muscle layer of the guinea pig ileum, a relatively radioresistant tissue. Single doses (up to 50 Gy) reduced the amount of sarcoplasmatic reticulum and condensed the myofibrils, as shown by electron microscopy 3 days post-irradiation. After that, contractility of smooth muscle strips was reduced. Ca(2+) handling was altered after irradiation, as shown in fura-2 loaded cells, with elevated basal intracellular Ca(2+), reduced amount of intrareticular Ca(2+), and reduced capacitive Ca(2+) entry. Radiation also induced apoptosis, judged from flow cytometry of cells loaded with proprium iodide. Electron microscopy showed that radiation caused condensation of chromatin in dense masses around the nuclear envelope, the presence of apoptotic bodies, fragmentation of the nucleus, detachment of cells from their neighbors, and reductions in cell volume. Radiation also caused activation of caspase 12. Apoptosis was reduced by the administration of the caspase inhibitor Z-Val-Ala-Asp-fluoromethyl-ketone methyl ester (Z-VAD-FMK) during the 3 day period after irradiation, and by the chelator of intracellular Ca(2+), 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), from 1 h before until 2 h after irradiation. BAPTA also reduced the effects of radiation on contractility, basal intracellular Ca(2+), amount of intrareticular Ca(2+), capacitative Ca(2+) entry, and apoptosis. In conclusion, the effects of gamma radiation on contractility, Ca(2+) handling, and apoptosis appear due to a toxic action of intracellular Ca(2+). Ca(2+)-induced damage to the sarcoplasmatic reticulum seems a key event in impaired Ca(2+) handling and apoptosis induced by gamma-radiation.

Morphologic and clinical effects of subretinal injection of indocyanine green and infracyanine green in rabbits.

PURPOSE: The aim of this study was to determine the effects of subretinal injection of indocyanine green (ICG), infracyanine (IfCG), and balanced salt solution (BSS) in rabbits. METHODS: Ten (10) animals were subjected to a subretinal injection of 0.05% ICG (279 mOsm), 0.5% IfCG (276 mOsm), and BSS (300 mOsm) used as a control. Animals were examined at 6, 12, and 24 h and 14 days following the surgical procedure by indirect binocular ophthalmoscopy, fluorescein angiography (FA), and light and transmission electron microscopy. RESULTS: The subretinal injection of ICG caused damage to all retinal layers and retinal pigment epithelium (RPE) during the entire follow-up. Subretinal injection of IfCG resulted in abnormalities of the photoreceptor outer segments (POSs) during the entire follow-up; however, abnormalities of the photoreceptor inner segments (PISs) and outer nuclear layer (ONL) were observed only 24 h and 14 days after surgery; no RPE damage was observed. FA showed that window defects were more prominent in the subretinal ICG bleb position than the IfCG-related area. BSS caused only abnormalities of the POS layer and no RPE alterations. CONCLUSIONS: Subretinal injection of 0.05% ICG results in more significant retinal damage than 0.5% IfCG. In this model, iodine-free IfCG demonstrates a safer profile than a tenfold lower concentration of ICG, which contains iodine in its composition.

Paracoccin, an N-acetyl-glucosamine-binding lectin of Paracoccidioides brasiliensis, is involved in fungal growth.

Paracoccin is an N-acetyl-glucosamine-binding lectin from Paracoccidioides brasiliensis, which can be obtained in small amounts either from culture supernatants or yeast cell extracts. In the present work, immunoelectron microscopy with mouse anti-paracoccin IgG localized the antigen to the cell wall of P. brasiliensis yeast forms. Paracoccin interacted with chitin, and colocalized with beta-1,4-homopolymer of GlcNAc to the budding sites of P. brasiliensis yeast cell. In order to evaluate the role of paracoccin on fungal growth, yeast cells were cultivated in the presence of anti-paracoccin antibodies. A significant reduction of both colony forming units and individual yeast cells was observed as well as morphological alterations such as smaller colonies and cells more loosely aggregated than in control cultures without the antibody. A role of paracoccin on the cell wall organization was reinforced by alterations in the labeling pattern of chitin when yeasts were treated with anti-paracoccin antibodies. Binding of specific antibodies to paracoccin may disrupt the paracoccin/chitin interactions, resulting in the inhibition of P. brasiliensis growth.

Morphological events during the Trypanosoma cruzi cell cycle.

The replication and segregation of organelles producing two identical daughter cells must be precisely controlled during the cell cycle progression of eukaryotes. In kinetoplastid flagellated protozoa, this includes the duplication of the single mitochondrion containing a network of DNA, known as the kinetoplast, and a flagellum that grows from a cytoplasmic basal body through the flagellar pocket compartment before emerging from the cell. Here, we show the morphological events and the timing of these events during the cell cycle of the epimastigote form of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease. DNA staining, flagellum labeling, bromodeoxyuridine incorporation, and ultra-thin serial sections show that nuclear replication takes 10% of the whole cell cycle time. In the middle of the G2 stage, the new flagellum emerges from the flagellar pocket and grows unattached to the cell body. While the new flagellum is still short, the kinetoplast segregates and mitosis occurs. The new flagellum reaches its final size during cytokinesis when a new cell body is formed. These precisely coordinated cell cycle events conserve the epimastigote morphology with a single nucleus, a single kinetoplast, and a single flagellum status of the interphasic cell.

Effects of subretinal injections of indocyanine green, trypan blue, and glucose in rabbit eyes.

PURPOSE: To evaluate the effects of subretinal injections of indocyanine green (ICG), trypan blue, glucose (GL), and balanced salt solution (BSS) in rabbits. DESIGN: Experimental study. PARTICIPANTS: Twenty Dutch-belted rabbits. METHODS: Ten animals underwent vitrectomy and subretinal injection of 0.02 ml of either 0.05% ICG (279 milliosmoles [mOsm]), 0.15% trypan blue (312 mOsm), 5% GL (280 mOsm), or BSS (300 mOsm), which was tested as a control. Ten additional animals underwent subretinal injection of 0.02 ml of 0.046% ICG (251 mOsm), 0.13% trypan blue (260 mOsm), 4.6% GL (253 mOsm), or BSS (300 mOsm). Animals were examined 6, 12, and 24 hours and 14 days after the procedure by fluorescein angiography and fundus evaluation; histologic studies were performed by light and transmission electron microscopy. MAIN OUTCOME MEASURES: Clinical outcome, fluorescein angiography, and histopathologic results. RESULTS: All subretinal blebs were flat 24 hours after the procedure. Fluorescein angiography showed window defects where ICG and trypan blue had been injected. Subretinal BSS and GL resulted in minimal abnormalities of the photoreceptor outer segments (POS) during follow-up. Hypo-osmolar GL caused edema in all retinal layers; pyknosis of the outer nuclear layer (ONL) was observed 24 hours after injection. Subretinal injection of trypan blue resulted in histologic abnormalities 24 hours and 14 days after surgery. Hypo-osmolar trypan blue caused edema of the POS and the photoreceptor inner segments and pyknosis of the ONL 6 and 12 hours after surgery; the retinal pigment epithelium also was affected 24 hours and 14 days after surgery. Subretinal injection of iso-osmolar and hypo-osmolar ICG caused severe damage of all retinal layers during the entire follow-up. CONCLUSIONS: Subretinal injection of 0.05% ICG results in more substantial retinal damage than that associated with the 0.15% trypan blue subretinal injection. The damage induced by hypo-osmolar solutions was more important than that caused by the iso-osmolar solutions. These findings emphasize that care must be taken regarding the solution osmolarity and that subretinal migration of these substances should be avoided during macular hole surgery.

Effects of subretinal injection of patent blue and trypan blue in rabbits.

PURPOSE: To investigate the histologic and clinical effects of subretinal injection of patent blue (PB) and trypan blue (TB) in rabbits. METHODS: Dutch-belted rabbits (n=8) were vitrectomized followed by subretinal injection of 2.4 mg/ml PB (285 mOsm) and 1.5 mg/ml TB (312 mOsm); balanced salt solution (BSS) (300 mOsm) served as the control. Animals were examined 6, 12, and 24 hr and 14 days after the procedure by fluorescein angiography (FA) and indirect ophthalmoscopy; for retinal toxicity, histologic evaluation studies were performed by light and transmission electron microscopy. RESULTS: FA examination demonstrated window defects suggestive of retinal pigment epithelium (RPE) atrophy in positions of subretinal TB injection, but this was not observed after subretinal injection of PB or BSS. Histologic evaluation disclosed only minimal abnormalities on the photoreceptor outer segment (POS) after subretinal injection of BSS during all follow-up. Subretinal injection of PB caused POS and photoreceptor inner segment (PIS) abnormalities 12 and 24 hr after surgery as well as outer nuclear layer (ONL) damage 14 days after surgery. Subretinal TB injection resulted in POS and PIS damage at 12 hr follow-up. The ONL damage was observed 24 hr after surgery; additionally, POS, PIS, ONL, and RPE abnormalities were observed 14 days after surgery after TB injection. CONCLUSIONS: Subretinal injection of TB induced more significant clinical and histologic damage of neurosensory retina/RPE than did PB or BSS. Future human studies are necessary to access the clinical relevance of these in vivo experiments.

Lectin KM+-induced neutrophil haptotaxis involves binding to laminin.

The lectin KM+ from Artocarpus integrifolia, also known as artocarpin, induces neutrophil migration by haptotaxis. The interactions of KM+ with both the extracellular matrix (ECM) and neutrophils depend on the lectin ability to recognize mannose-containing glycans. Here, we report the binding of KM+ to laminin and demonstrate that this interaction potentiates the KM+-induced neutrophil migration. Labeling of lung tissue by KM+ located its ligands on the endothelial cells, in the basement membrane, in the alveolus, and in the interstitial connective tissue. Such labeling was inhibited by 400 mM D-mannose, 10 mM Manalpha1-3[Manalpha1-6]Man or 10 microM peroxidase (a glycoprotein-containing mannosyl heptasaccharide). Laminin is a tissue ligand for KM+, since both KM+ and anti-laminin antibodies not only reacted with the same high molecular mass components of a lung extract, but also determined colocalized labeling in basement membranes of the lung tissue. The relevance of the KM+-laminin interaction to the KM+ property of inducing neutrophil migration was evaluated. The inability of low concentrations of soluble KM+ to induce human neutrophil migration was reversed by coating the microchamber filter with laminin. So, the interaction of KM+ with laminin promotes the formation of a substrate-bound KM+ gradient that is able to induce neutrophil haptotaxis.

Granuloma formation in vitro requires B-1 cells and is modulated by Paracoccidioides brasiliensis gp43 antigen.

The mechanisms that determine granuloma formation and the significance of this type of inflammatory response in the pathogenesis of fungal diseases such as paracoccidioidomycosis are far from fully understood. We developed a granuloma model in vitro using beads to evaluate the role of isolated mouse peritoneal macrophages and B-1 cells. We also investigated granuloma formation in the presence of gp43, the main antigenic component of Paracoccidioides brasiliensis, which is secreted exocellularly. To determine whether B-1 cells, macrophages, or both, participate in granuloma formation, peritoneal cells from Xid mice, which lack B-1 cells, were used. Granuloma-like structures were not formed with Xid peritoneal cells or with cells from wild type mice that had their peritoneal and pleural cavities irradiated before the cultures were established. Granulomas were observed either when total adherent peritoneal cells or when isolated B-1 cells were added to macrophage cultures. The data strongly suggest that an interaction of B-1 cells and macrophages plays an important role in granuloma-like formation in this experimental model and that the presence of gp43 strongly stimulates this response.

Elastoderma of the neck in a teenage boy.

Elastoderma is an exceedingly rare condition characterized clinically as an acquired localized laxity of skin and, histologically, as an excessive accumulation of pleomorphic elastic structures within the dermis. We report the case of a 16-year-old white man with a 2-year history of progressive laxity, extensibility, and wrinkling of the skin localized to the anterior aspect of the neck. Histologic examination of specimens from the affected area showed dense aggregates of eosinophilic material present within the dermis. Transmission and scanning electron microscopic examination demonstrated apposition of abnormal elastic structures at the periphery of elastic tissue fibers, with grapelike globular structures. To our knowledge, we report the third case of elastoderma.

Replacement of Asymmetric Synaptic Profiles in the Molecular Layer of Dentate Gyrus Following Cycloheximide in the Pilocarpine Model in Rats.

Mossy fiber sprouting is among the best-studied forms of post-lesional synaptic plasticity and is regarded by many as contributory to seizures in both humans and animal models of epilepsy. It is not known whether mossy fiber sprouting increases the number of synapses in the molecular layer or merely replaces lost contacts. Using the pilocarpine (Pilo) model of status epilepticus to induce mossy fiber sprouting, and cycloheximide (CHX) to block this sprouting, we evaluated at the ultrastructural level the number and type of asymmetric synaptic contacts in the molecular layer of the dentate gyrus. As expected, whereas Pilo-treated rats had dense silver grain deposits in the inner molecular layer (IML) (reflecting mossy fiber sprouting), pilocarpine + cycloheximide (CHX + Pilo)-treated animals did not differ from controls. Both groups of treated rats (Pilo group and CHX + Pilo group) had reduced density of asymmetric synaptic profiles (putative excitatory synaptic contacts), which was greater for CHX-treated animals. For both treated groups, the loss of excitatory synaptic contacts was even greater in the outer molecular layer than in the best-studied IML (in which mossy fiber sprouting occurs). These results indicate that mossy fiber sprouting tends to replace lost synaptic contacts rather than increase the absolute number of contacts. We speculate that the overall result is more consistent with restored rather than with increased excitability.

Mouse resident peritoneal macrophages partially control in vitro infection with Coxiella burnetii phase II.

Coxiella burnetii, the agent of Q fever in man and of coxiellosis in other species, is a small, dimorphic, obligate intracellular bacterium, sheltered within large, acidified, and hydrolase-rich phagosomes. Although several primary and established cell lines, macrophage-like cells, and primary macrophages from other species have been infected with C. burnetii, the infection of mouse primary macrophages has not been sufficiently characterized. In this report quantification of DAPI (4', 6-diamino-2-phenylindole) fluorescence images acquired by confocal microscopy, and transmission electron microscopy were used to compare the infection of three mouse-derived cells, L929 fibroblasts, J774 macrophage-like cells, and resident peritoneal macrophages, with a phase II clone of C. burnetii known to be non-virulent for mammals. Infected peritoneal phagocytes differed from L929 or J774 cells in that: (a) large vacuoles took longer to appear (3-5 d instead of 2), and were only found in a subset (20-30%) of macrophages, as opposed to in more than 70% of the other cells; (b) total and vacuole-associated relative bacterial loads in L929 and J774 cells were several-fold higher than in peritoneal macrophages; (c) estimated doubling times of the bacteria were about 68 h in the primary macrophages, 18 h in J774 and 22 h in L929 cells. Thus, mouse resident peritoneal macrophages control both the formation of the large vacuoles and the intracellular proliferation of C. burnetii phase II.