Interstitial Axon Collaterals of Callosal Neurons Form Association Projections from the Primary Somatosensory to Motor Cortex in Mice.

Association projections from cortical pyramidal neurons connect disparate intrahemispheric cortical areas, which are implicated in higher cortical functions. The underlying developmental processes of these association projections, especially the initial phase before reaching the target areas, remain unknown. To visualize developing axons of individual neurons with association projections in the mouse neocortex, we devised a sparse labeling method that combined in utero electroporation and confocal imaging of flattened and optically cleared cortices. Using the promoter of an established callosal neuron marker gene that was expressed in over 80% of L2/3 neurons in the primary somatosensory cortex (S1) that project to the primary motor cortex (M1), we found that an association projection of a single neuron was the longest among the interstitial collaterals that branched out in L5 from the earlier-extended callosal projection. Collaterals to M1 elongated primarily within the cortical gray matter with little branching before reaching the target. Our results suggest that dual-projection neurons in S1 make a significant fraction of the association projections to M1, supporting the directed guidance mechanism in long-range corticocortical circuit formation over random projections followed by specific pruning.

Kcnab1 Is Expressed in Subplate Neurons With Unilateral Long-Range Inter-Areal Projections.

Subplate (SP) neurons are among the earliest-born neurons in the cerebral cortex and heterogeneous in terms of gene expression. SP neurons consist mainly of projection neurons, which begin to extend their axons to specific target areas very early during development. However, the relationships between axon projection and gene expression patterns of the SP neurons, and their remnant layer 6b (L6b) neurons, are largely unknown. In this study, we analyzed the corticocortical projections of L6b/SP neurons in the mouse cortex and searched for a marker gene expressed in L6b/SP neurons that have ipsilateral inter-areal projections. Retrograde tracing experiments demonstrated that L6b/SP neurons in the primary somatosensory cortex (S1) projected to the primary motor cortex (M1) within the same cortical hemisphere at postnatal day (PD) 2 but did not show any callosal projection. This unilateral projection pattern persisted into adulthood. Our microarray analysis identified the gene encoding a ß subunit of voltage-gated potassium channel (Kcnab1) as being expressed in L6b/SP. Double labeling with retrograde tracing and in situ hybridization demonstrated that Kcnab1 was expressed in the unilaterally-projecting neurons in L6b/SP. Embryonic expression was specifically detected in the SP as early as embryonic day (E) 14.5, shortly after the emergence of SP. Double immunostaining experiments revealed different degrees of co-expression of the protein product Kvß1 with L6b/SP markers Ctgf (88%), Cplx3 (79%), and Nurr1 (58%), suggesting molecular subdivision of unilaterally-projecting L6b/SP neurons. In addition to expression in L6b/SP, scattered expression of Kcnab1 was observed during postnatal stages without layer specificity. Among splicing variants with three alternative first exons, the variant 1.1 explained all the cortical expression mentioned in this study. Together, our data suggest that L6b/SP neurons have corticocortical projections and Kcnab1 expression defines a subpopulation of L6b/SP neurons with a unilateral inter-areal projection.

Frontal hypoperfusion in depressed patients with dementia of Alzheimer type demonstrated on 3DSRT.

AIMS: Depressive symptoms are common in patients with dementia of Alzheimer type (DAT) and contribute to clinical morbidity. Previous studies have suggested that hypoperfusion in the prefrontal cortex and anterior cingulate gyrus are involved in the pathophysiology of depression in DAT. Using 3-D stereotactic region of interest (ROI) template (3DSRT), fully automated ROI analysis software, the purpose of the present study was to investigate the relationship between depressive symptoms and regional cerebral blood flow (rCBF) in DAT. METHODS: Technetium-99m-ethyl cysteinate dimer ((99m)Tc-ECD) single-photon emission computed tomography (SPECT) and Japanese version of the Neuropsychiatric Inventory (NPI) were carried out in 35 patients diagnosed as having mild-moderate DAT according to DSM-IV. These patients were divided into the depressive group (D group: n = 17) and non-depressive group (ND group: n = 18) using the NPI depression items. All data from SPECT images were analyzed using 3DSRT software. On 3DSRT the perfusion ratios (rCBF of bilateral callosomarginal, precentral, central, parietal, angular, temporal, posterior cerebral, pericallosal, lenticular nucleus, thalamus and hippocampus/cerebellar hemisphere) of each segment were compared between the D group and the ND group. RESULTS: The perfusion ratios of the left callosomarginal segment for the D group were significantly lower (P < 0.05) than those of the ND group. CONCLUSIONS: Hypoperfusion in the left frontal cortex contributes to the expression of depressive symptoms in patients with DAT.

The relationship between depressive symptoms and prefrontal hypoperfusion demonstrated by eZIS in patients with DAT.

Depressive symptoms are common in Alzheimer's disease (AD) and contribute to clinical morbidity. Previous studies have suggested that hypoperfusion in the prefrontal cortex and anterior cingulate gyrus are involved in the pathophysiology of depression. Using the easy Z-score imaging system (eZIS), we investigated the relationship between depressive symptoms and prefrontal hypoperfusion in AD. Tc-99m-ethyl cysteinate dimer (Tc-99m-ECD)-single photon emission tomography (SPECT) and Neuropsychiatric Inventory (NPI) were performed in forty-four patients diagnosed as having Dementia of Alzheimer's type (DAT) with the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). These patients were divided into the depressive group (D group: n=26) and non-depressive group (ND group: n=18) using NPI depression items. All data from SPECT images were analyzed using eZIS software. Scores in four regions were determined by Z-values; these regions consisted of each side of the prefrontal cortex and anterior cingulate gyrus. The mean scores between the D group and ND group were compared. The mean scores of the left prefrontal cortex in the D group were significantly higher (p<0.0125) than those in the ND group. There were no significant differences in the scores of the right prefrontal cortex and the bilateral anterior cingulate gyrus between these two groups (Mann-Whitney U-test). These findings suggest that hypoperfusion in the left prefrontal area contributes to the expression of depressive symptoms in patients with DAT.

3DSRT evaluation of responses of Alzheimer type dementia to donepezil hydrochloride therapy.

BACKGROUND: Dementia of Alzheimer type (DAT) has been diagnosed objectively by using single photon emission tomography (SPECT). Donepezil hydrochloride (donepezil) is available for the symptomatic treatment of DAT. In a quantitative evaluation of therapeutic response in DAT, to compare with regional cerebral blood flows (rCBF) of various lesions before and after treatment, uptake in some sorts of cerebral regions of interests (ROIs) were used to be measured. But ROI analysis has problems such as poor reproducibility and lack of objectivity. The aim of this study was to investigate the evaluation of therapeutic response by three-dimensional stereotaxic ROI template (3DSRT), fully automated ROI analysis software, which can objectively estimate rCBF. METHODS: SPECT studies and Alzheimer's Disease Assessment Scale Japan cognitive Subscale function test ADAS-Jcog, as recognitive function test were performed for 22 patients (16 females, 6 males mean age = 73.6 years) who were diagnosed as DAT. On 3DSRT, we compared ratios of the rCBF values of the parietal lobes, temporo-occipital lobes, hippocampus, corpus callosum and the frontal lobes/cerebellar hemispheres before and after medical treatment. To determine a cut-off number of areas exhibiting improved blood flow optimal as an indicator of improvement in cognitive function in response to treatment, receiver operating characteristics (ROC) analysis of number of areas improved blood flow was performed. RESULTS: The number of cases exhibiting changes in cognitive function was greatest when the cut-off number of areas exhibiting improved blood flow was set at 5. CONCLUSIONS: The possibility of evaluation of therapeutic response to Donepezil in patients with DAT using 3DSRT was thus demonstrated by our study.

The association between Staphylococcus aureus strains carrying panton-valentine leukocidin genes and the development of deep-seated follicular infection.

BACKGROUND: Panton-Valentine leukocidin (PVL) is mainly associated with necrotic suppurative lesions, such as furuncles and abscesses in the skin and subcutaneous tissue, but it has also been isolated from patients with community-acquired, severe, necrotizing pneumonia. However, the clinical manifestations of furuncles caused by PVL-producing Staphylococcus aureus and the role of patients' background are not fully understood. METHODS: We used polymerase chain reaction amplification to test for the PVL gene in 161 strains of S. aureus isolated from suppurative skin lesions. For all PVL gene-positive strains isolated from furuncles, we analyzed cutaneous manifestations, patient background characteristics, and bacteriological markers, including coagulase types, presence of the mecA gene, and toxin profiles, and we compared these results with those for PVL gene-negative strains. RESULTS: PVL genes were detected in 16 (40%) of the 40 S. aureus strains isolated from furuncles, 2 (28%) of the 7 strains isolated from carbuncles, 1 (14%) of the 7 strains isolated from abscesses, and 1 (5%) of the 20 strains isolated from folliculitis. PVL gene-positive S. aureus usually causes multiple (rather than single) furuncles, and such furuncles are usually associated with more-intense erythema around the lesions. PVL gene-positive strains were isolated from young adults without underlying diseases, whereas PVL gene-negative strains were isolated from patients with various systemic complications, including diabetes, leukemia, and autoimmune diseases. CONCLUSIONS: PVL gene-positive S. aureus strains are involved in the development of multiple furuncles with more-intense erythema, particularly in healthy young adults. An understanding of the characteristics of furuncles due to PVL gene-positive strains might be useful for preventing the development of the severe systemic infections.

Assessment of cadexomer iodine against Staphylococcus aureus biofilm in vivo and in vitro using confocal laser scanning microscopy.

Cadexomer iodine releases iodine (0.9% weight/weight) slowly from beads of dextrin and epichlorhydrin. This preparation is an effective debridement and antiseptic agent for chronic exdudative wounds. The purpose of the present study is to examine the influence of cadexomer iodine against glycocalyx production of Staphylococcus aureus isolated from furuncle lesions on cut wounds in mice using confocal laser scanning microscope (CLSM), and the increase in and glycocalyx production of S. aureus in vitro. In the present study, distinct S. aureus cells and glycocalyx were not detected in the dermis around the cadexomer iodine beads or within those beads, while S. aureus cells encircled by glycocalyx were soaked up by the cadexomer beads and were detected within them in vivo and in vitro. We suggest that cadexomer iodine soaks up S. aureus cells encircled by glycocalyx, directly destroys biofilm structures, and collapses glycocalyx during dehydration, and further, that iodine can subsequently kill S. aureus cells within biofilm. Cadexomer iodine is a promising treatment to clear S. aureus cells within biofilm from skin lesions of exudative or infectious wounds and to prevent wound exacerbation.

Assessment of Streptococcus pyogenes microcolony formation in infected skin by confocal laser scanning microscopy.

BACKGROUND: Streptococcus pyogenes and Staphylococcus aureus are often simultaneously detected from many cases of non-bullous impetigo with atopic dermatitis. OBJECTIVES: Using confocal laser scanning microscopy (CLSM), to investigate formation of S. pyogenes microcolonies in skin lesions. METHODS: The S. pyogenes cells in the stationary growth phase alone were strongly stained with fluorescein isothiocyanate-concanavalin A (FITC-ConA), and this staining was reduced by pretreatment with amylase. Although the components of sugars in glycocalyx produced by S. pyogenes cells are unknown, we suggested that the materials stained by FITC-ConA were consistent with the presence of ConA-reactive sugars in glycocalyx produced by S. pyogenes cells. RESULTS: S. pyogenes cells associated with streptococcal impetigo skin and croton-oil inflamed mouse skin formed microcolonies encircled by materials (glycocalyx) that stained strongly with FITC-ConA, and these findings were consistent with those in biofilms. In croton-oil inflamed mouse skin, polymorphonuclear leukocytes (PMNs) infiltrated to just below the epidermis in the cefdinir-treated group but only to the middle dermis in the cefdinir-non-treated group. In this case S. pyogenes and S. aureus cells formed separate microcolonies and existed independently in the outer walls of pustule lesions of streptococcal impetigo. CONCLUSION: In skin infections, S. pyogenes and S. aureus formed aggregates of microcolonies (similar to that in biofilms) encircled by glycocalyx, which can make the infection hard to eradicate using an antimicrobial agent alone. The effect of conventional antimicrobial agents against biofilm is mainly due to the increase of the invasion of PMNs into the biofilm.

Actions of gluco-oligosaccharide against Staphylococcus aureus.

Heavy colonization of atopic dermatitis (AD) with Staphylococcus aureus is well documented. The purpose of the present study is to examine the actions of gluco-oligosaccharide (G-OS) against S. aureus for the control of AD skin lesions infected with S. aureus. The colony counts of S. aureus cells in 0.5% sodium chloride solution supplemented with 5% G-OS (pH 4.8) were about 10-fold lower than those in 0.5% sodium chloride solution (pH 6.6; control) after incubation for 24 hours. The colony counts of S. aureus cells attached on the coverslips (pre-treatment with 1% and 5% G-OS/PBS and following treatment with plasma) were about 10-fold lower than those on the coverslips (pre-treatment with PBS and following treatment with plasma; control) in PBS after incubation for 24 hours. The materials (sugars, probably glycocalyx) that stained positively for fluorescein-isothiocyanate (FITC) -concanavalin A and were consistent with the presence of S. aureus cells were reduced when S. aureus cells attached to the coverslips treated with 5% GC-OS. In conclusion, C-OS is a promising agent that can be applied topically in a cream to clear adherent S. aureus cells from skin lesions of AD in order to prevent its exacerbation. Further, 5% C-OS can inhibit glycocalyx production by S. aureus cells and consequently have some suppressive effect on the colonization of S. aureus on the horny cells of AD lesions.

Dynamic alteration of human beta-defensin 2 localization from cytoplasm to intercellular space in psoriatic skin.

Defensins are cationic antimicrobial peptides with a broad spectrum. Recently human beta-defensin 2 (hBD-2) has been isolated from psoriatic skin; however, its exact localization and fate have not been fully understood. We studied the distribution pattern of hBD-2 in skin tissues of psoriasis and other inflammatory skin diseases. In the upper spinous and granular layer of psoriasis vulgaris hBD-2 was present in the cytoplasm. In the horny layer the positive signals were in a basket-weave pattern, indicating possible accumulation of hBD-2 in the intercellular space. The similar pattern of hBD-2 distribution was observed in the lesions of nummular eczema and atopic dermatitis. hBD-2 was not detected in the section of normal elbow and knee skin. When isolated psoriatic scales were stained, hBD-2 was detected in a wrapping paper-like distribution pattern surrounding the corneocytes. In horny layer of psoriatic skin hBD-2 was closely associated or colocalized with elafin, which is known to be in extracellular space, as demonstrated by double staining. Western blot analysis using cultured human keratinocytes detected hBD-2 with an expected size in the conditioned medium and in the cell lysates when stimulated with 5% FCS or IL-alpha. These results indicate that hBD-2 was synthesized and remained in cytoplasm in the upper spinous and granular layer, and then secreted into intercellular space in the horny layer. This dynamic change in hBD-2 distribution in epidermis is certainly relevant to function as an innate host defense mechanism against invading micro-organisms.

Actions of farnesol and xylitol against Staphylococcus aureus.

BACKGROUND: Heavy colonization of atopic dermatitis (AD) with Staphylococcus aureus is well documented. The isolation rate of methicillin-resistant S. aureus is high in strains from AD in Japan. Our objective in the present study was to investigate the actions of farnesol and xylitol against S. aureus for the control of AD skin lesion-colonizing S. aureus. METHODS: We examined the actions of farnesol on plasma coagulation and superantigenic exotoxin production by S. aureus, the antimicrobial activity of beta-lactam antibiotics combined with farnesol at concentrations below the minimal inhibitory concentration (MIC) and the effect of xylitol on glycocalyx production. RESULTS: Coagulation by S. aureus cells was inhibited in plasma containing farnesol at a concentration of 1/12 of the MIC (100 microg/ml) after incubation for 24 h. The production of superantigenic exotoxins by S. aureus cells with farnesol (100 microg/ml) was about 10 times lower than that by S. aureus cells alone. The MICs of ampicillin and cefdinir against S. aureus were reduced to < or =0.06 microg/ml in Mueller-Hinton agar plates with farnesol (100 microg/ml). We suggest that farnesol at concentrations above the MIC had a suppressive effect against S. aureus cells in the exponential and stationary phase and acted on the cell wall of S. aureus cells in both phases. CONCLUSIONS: Farnesol is a promising adjuvant agent against S. aureus skin infections treated with beta-lactam antibiotics. Further, 5% xylitol inhibited glycocalyx production by S. aureus cells and consequently had a suppressive effect on the colonization of S. aureus on the horny cells of AD lesions.

Effects of human neutrophil peptide-1 on the expression of interstitial collagenase and type I collagen in human dermal fibroblasts.

Human neutrophil peptide-1 (HNP-1), a defensin with antimicrobial properties, is also thought to promote wound healing. To elucidate the mechanism by which wound healing is facilitated by this factor, we investigated the effect of HNP-1 on the expression of interstitial collagenase (matrix metalloproteinase 1, MMP-1), collagen types I and III, and tissue inhibitor of metalloproteinase 1 (TIMP-1) by cultured fibroblasts by means of RT-PCR and ELISA. Our results showed that synthetic HNP-1 increased the expression of proalpha1(I) collagen mRNA and protein. In contrast, the expression of MMP-1 was decreased at both the mRNA and protein levels. Our observations suggest that HNP-1 may promote wound repair by enhancing extracellular matrix deposition and by controlling its degradation.

Confocal laser microscopic observation of glycocalyx production by Staphylococcus aureus in vitro.

We used a scanning confocal laser microscope to study the effects of various agents on sugar production by Staphylococcus aureus in vitro. S. aureus cells attached to coverslips in Pl-TSB (plasma:tryptic soy broth=1:1) were stained with fluorescein isothiocyanate-conjugated concanavalin A (FITC-conA) and were more strongly stained over time. We considered that the materials that stained positive for FITC-conA consistent with S. aureus cells were sugars, probably glycocalyx, produced by the S. aureus cells. Since the cells in the stationary growth phase alone were strongly stained with FITC-conA, all S. aureus cells attached to the coverslips in Pl-TSB were considered to be in this phase (low growth rate). The positive staining for FITC-conA was markedly reduced when fibrin was not formed in Pl-TSB with plasmin and sucrose, and was also markedly reduced when the fibrin in Pl-TSB was destroyed with plasmin. In conclusion, the results of the present study indicate that the existence of fibrin is essential for glycocalyx production and biofilm formation of S. aureus cells to aid in the attachment of S. aureus cells in vitro, because S. aureus cells attached on coverslips and fibrin alone produce glycocalyx. Of the antimicrobial agents tested, sulfadiazine silver most strongly inhibited the production of FITC-conA-positive materials by S. aureus cells at a sub-MIC concentration. Plasmin, sucrose, and sulfadiazine silver may be useful topical applications for use on clinical dermatology for the prevention and the treatment of staphylococcal biofilms. We consider that this simple method is very useful for the detection of S. aureus glycocalyx on dermatology field.

Comparison of the severity of atopic dermatitis lesions and the density of Staphylococcus aureus on the lesions after antistaphylococcal treatment.

We examined the relationship between atopic dermatitis (AD) and Staphylococcus aureus by comparing changes in AD lesions and the bacterial density on the lesions after antimicrobial treatment with cefdinir. We found that there was a greater density of S. aureus on red erythemas and exudative lesions than in light/dark red erythemas and non-exudative lesions of AD. Forty-one of 59 cases (69%) showed a decrease in colony count following antimicrobial treatment. In 28 of 39 cases (72%) there was a decrease of erythema, and in 18 of 22 cases (82%) there was a decrease in the amount of exudate both associated with a decrease in colony density following antimicrobial treatment. Because acute phases of atopic dermatitis, such as red erythemas and exudative lesions, were closely related to the colonization of S. aureus, dense colonization with S. aureus may be an important factor in the exacerbation of AD. We believe that staphylococcal products such as α-toxin, various enzymes, coagulase, and superantigenic exotoxins affect some aspect of the inflammatory process, resulting in exacerbation of AD. J Infect Chemother 1996;2:70-74.