Simple, rapid, and reproducible capillary gel electrophoresis separation and laser-induced fluorescence detection of DNA topoisomers with unmodified fused silica separation capillaries.

The topology of DNA is a critical quality attribute for plasmid-based pharmaceuticals, making quantification of trace levels of plasmid topoisomers an important analytical priority. An automated and cost-effective method based on capillary gel electrophoresis laser-induced fluorescence detection is described. The method outlined in this report is significant because it is easily implemented by any laboratory for which routine analyses of plasmid topology are critical for the development of new plasmid-based therapies as well as for quality control of gene therapies utilizing supercoiled DNA. Detection of topoisomers was achieved by incorporating ethidium bromide in the separation medium. The detector response was improved by 3 orders of magnitude by utilizing a 605-nm optical filter with a 15-nm bandwidth. Separations of linear, open circle, supercoiled, and multimer DNA plasmids ranging from 4.2 to 10.5 kbp were accomplished in under 6 min using an unmodified fused silica capillary (50-µm internal diameter). The background electrolyte was comprised of 0.5% gel, which was hydroxypropylmethyl cellulose, 1 mM ethylenediaminetetraacetic acid, and 50 mM N-(2-acetamido)-2-aminoethanesulfonic acid (pH of 6.25). The separations, which balanced the bulk electroosmotic flow, the electrophoretic mobility of the DNA, and gel sieving were dependent upon the pH of the electrolyte and the gel concentration. Reproducibility was dependent upon the procedure used to prepare the gel as well as other factors including the ethidium bromide concentration and capillary conditioning. A single unmodified capillary operated for more than 150 runs had an across-day migration time precision of 1% relative standard deviation and percent area precision of 10% relative standard deviation.

Daptomycin: A Novel Macrocyclic Antibiotic as a Chiral Selector in an Organic Polymer Monolithic Capillary for the Enantioselective Analysis of a Set of Pharmaceuticals.

Daptomycin, a macrocyclic antibiotic, is here used as a new chiral selector in preparation of chiral stationary phase (CSP) in a recently prepared polymer monolithic capillary. The latter is prepared using the copolymerization of the monomers glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) in the presence of daptomycin in water. Under reversed phase conditions (RP), the prepared capillaries were tested for the enantioselective nanoliquid chromatographic separation of fifty of the racemic drugs of different pharmacological groups, such as adrenergic blockers, H1-blockers, NSAIDs, antifungal drugs, and others. Baseline separation was attained for many drugs under RP-HPLC. Daptomycin expands the horizon of chiral selectors in HPLC.

Two new reliable immunohistochemical methods for simultaneous identification of capillaries, the three types of fibers and basal lamina in human skeletal muscle.

Capillary network of skeletal muscle has a crucial role in oxygen supply and is strongly associated with the phenotype and metabolic profile of muscle fibers. Abundant literature has explored capillarization of skeletal muscle in different populations and in response to different interventions. Capillary and fiber type identification techniques have considerably evolved over the last decades, but to the best of our knowledge, no validated immunohistochemical method has yet been developed to simultaneously identify capillaries (using CD31), the three different muscle fiber types, and basal lamina. Nine human muscle biopsies of vastus lateralis were stained using 5 different methods to test: the reliability of different CD31 antibodies for capillary identification, the reliability between single-section or serial-section methods, and the intra-experimenter reproducibility in visual detection of capillaries. High reliability for the different antibodies directed against capillaries was observed for capillary contacts (CC) measurements (intra-class correlations (ICC) [ICC95%] of 0.89 [0.72; 0.96] for type I fibers, 0.93 [0.81; 0.97] for type IIA fibers, 0.88 [0.71; 0.96] for type IIX fibers, 0.95 [0.86; 0.98] for all fiber types) as well as a high level of similarity between single and serial sections methods. A strong similarity in capillary analysis between the different methods was obtained for each sample measurements. Analysis of Lin's concordance correlation coefficients and Bland and Altman's graphics showed a strong intra-experimenter reproducibility. This article proposes two time- and tissue-sparing immunohistochemical methods to accurately assess a complete fiber typing (type I, IIA, and IIX) along with muscle capillarization on a single muscle section.

3D analysis of capillary network in skeletal muscle of obese insulin-resistant mice.

In obesity, the skeletal muscle capillary network regresses and the insulin-mediated capillary recruitment is impaired. However, it has been shown that in the early stage of advanced obesity, an increased functional vascular response can partially compensate for other mechanisms of insulin resistance. The present study aimed to investigate the changes in the capillary network around individual muscle fibres during the early stage of obesity and insulin resistance in mice using 3D analysis. Capillaries and muscle fibres of the gluteus maximus muscles of seven high-fat-diet-induced obese and insulin-resistant mice and seven age-matched lean healthy mice were immunofluorescently labelled in thick transverse muscle sections. Stacks of images were acquired using confocal microscope. Capillary network characteristics were estimated by methods of quantitative image analysis. Muscle fibre typing was performed by histochemical analysis of myosin heavy chain isoforms on thin serial sections of skeletal muscle. Capillary length per muscle fibre length and capillary length per muscle fibre surface were increased by 27% and 23%, respectively, around small muscle fibres in obese mice, while there were no significant comparative differences around large fibres of obese and lean mice. Furthermore, the capillarization was larger around small compared to large fibres and there was a shift toward fast type myosin heavy chain isoforms, with no significant changes in muscle fibre diameters, tortuosity and anisotropy in obese mice. Overall, the results show that obese insulin-resistant mice have selective increase in capillarization around small predominantly intermediate muscle fibres, which is most likely related to the impaired glucose metabolism characteristic of type 2 diabetes.

Diabetic Ketoacidosis in Adolescents and Children: A Prospective Study of Blood versus Urine Ketones in Monitoring Therapeutic Response.

BACKGROUND: diabetic ketoacidosis (DKA) is a potentially lethal complication of diabetes mellitus (DM). There is no study in Indonesia that compares the much-preferred capillary beta hydroxybutirate (ß-OHB) measurement to urine acetoacetate in monitoring therapeutic response of DKA in adolescents. METHODS: a prospective study of 37 adolescents and children with DKA in Cipto Mangunkusumo Hospital was done between June 2006 and March 2011. The patients were followed until the time of DKA resolution. Hourly measurement of random blood glucose, capillary ß-OHB concentration, and urine ketones were done, while blood gas analysis and electrolyte were measured every four hours. RESULTS: median time to resolution was 21 (9-52) hours. Compared to urine ketones, capillary ß-OHB concentration showed stronger correlation with pH (r= -0,52, p= 0,003 vs r= -0,49, p= 0,005) and bicarbonate level (r=-0,60, p=0.000 vs r= -0.48, p=0.007) during the median time of DKA resolution. All capillary ß-OHB measurement yielded negative results at median time of DKA resolution, while urine ketones were still detected up to 9 hours after resolution. CONCLUSION: blood ketone concentration showed better correlation with pH and bicarbonate level, as a tool to monitor therapeutic response in DKA in adolescent, compared to traditional urine ketones test in adolescents.

In vivo Imaging of the Cerebral Endothelial Glycocalyx in Mice.

BACKGROUND/AIMS: Endothelial glycocalyx refers to the proteoglycan or glycoprotein layer of vessel walls and has critical physiological functions. Cerebral glycocalyx may have additional functions considering the blood-brain barrier and other features. However, the assessment of it has only been performed ex vivo, which includes processes presumably damaging the glycocalyx layer. Here we visualize and characterize the cerebral endothelial glycocalyx in vivo. METHODS: We visualized and quantified the cerebral endothelial glycocalyx in vivo under a 2-photon microscope by tagging glycocalyx and vessel lumen with wheat germ agglutinin lectin and dextran, respectively. The radial intensity was analyzed to measure the thickness of the cerebral endothelial glycocalyx in each vessel type. RESULTS: Cerebral arteries and capillaries have an intact endothelial glycocalyx, but veins and venules do not. The thickness of the glycocalyx layer in pial arteries, penetrating arteries, and capillaries was different; however, it was not correlated with the vessel diameter within each vessel type. CONCLUSION: We characterized the distribution of the cerebral endothelial glycocalyx in vivo. Compared to the results from ex vivo studies, the layer is thicker, indicating that the layer may be damaged in ex vivo systems. We also observed an inhomogeneous cerebral endothelial glycocalyx distribution that might reflect the functional heterogeneity of the vessel type.

Plasma endothelial microparticles reflect the extent of capillaroscopic alterations and correlate with the severity of skin involvement in systemic sclerosis.

INTRODUCTION AND AIM: Endothelial microparticles (EMPs) are membrane-coated vesicles shed from endothelial cells and are considered markers of the endothelial state. It has been shown that total numbers of circulating EMPs are increased in patients with systemic sclerosis (SSc), but their clinical correlations have not yet been investigated in detail. We aimed to assess possible relationships between circulating EMPs and clinical as well as laboratory features among SSc patients with special attention to possible association with alteration in microvascular morphology objectified on nailfold videocapillaroscopy and clinical signs of microvascular complications. MATERIALS AND METHODS: The study included 47 SSc patients and 27 age- and sex-matched healthy controls. EMPs were identified with flow cytometry after staining platelet-poor plasma with combinations of fluorescent cell-specific monoclonal antibodies (anti-CD31, -51, -42b, -62E and Annexin V). The following types of EMPs were evaluated: total EMPs (CD31+/CD42b-), activated EMPs (CD62E+/AnnV-,) and apoptotic EMPs (CD62E+/AnnV+ or CD51+). Clinical evaluation of patients was obtained, including nailfold videocapillaroscopy. RESULTS: All types of EMPs were significantly elevated in SSc patients as compared with healthy controls. We found significant inverse correlation between severity of skin involvement and values of total EMPs (r=-0.32; p=0.02) and their levels tended to be lower in SSc patients with digital ulcers when compared to those without ischaemic skin lesions (p=0.09). Total EMPs and activated EMPs showed correlations with the number of ramified capillaries (r=-0.40 and r=0.37, respectively, p<0.05 for both). Moreover, total EMPs inversely correlated with the severity of capillary loss (r=-0.35, p<0.05) and their levels were significantly lower in patients with late NVC pattern with respect to those with early microangiopathy (p<0.05). On the other hand, active NVC pattern was characterized by strongly elevated levels of activated EMPs when compared to an early vascular alteration (p<0.05). CONCLUSIONS: Our results suggest that quantity and phenotype of circulating EMPs might indicate on molecular vascular damage with endothelial dysfunction and to reflect progressive loss of capillaries consequencing in microvascular insufficiency in SSc patients.

Proportion of daily capillary blood glucose readings required in the target range for target glycaemic control: shift of focus from target range to proportion in range.

AIM: Most guidelines provide people with Type 1 diabetes with pre- and post-meal capillary blood glucose (CBG) targets to achieve optimal glycaemic control. We evaluated the proportion of daily CBG readings between 4 and 10 mmol/l in people achieving different HbA1c levels. METHOD: We analysed CBG data from routine pump/meter downloads from 201 adults treated with continuous subcutaneous insulin infusion (CSII) at a single hospital clinic. Exclusion criteria were CSII < 6 months, < 3 CBG/day, pregnancy, haemoglobinopathy and continuous sensor use. People were categorized into three groups based on HbA1c : < 58 mmol/mol, < 7.5% (n = 58); 58-74 mmol/mol, 7.5-8.9% (n = 107); and ≥ 75 mmol/mol, ≥ 9.0% (n = 36). RESULTS: Participants had a mean age of 43 ± 13 years and mean HBA1c of 64 mmol/mol (8.0 ± 1.1%). 47% of people started CSII for raised HbA1c , 25% due to hypoglycaemia and the rest during pregnancy. Downloads contained a mean of 22 ± 6.8 days of data per participant. CBG frequency was similar between the three groups (5.6 ± 2.0, 5.6 ± 1.9 and 5.4 ± 1.2 CBG/day; P = 0.468). The proportion of CBG readings between 4 and 10 mmol/l (72-180 mg/dl) was 57.3 ± 25.4%, 50.6 ± 11.1% and 39.9 ± 16.5% (P < 0.0001); < 4 mmol was 13.8%, 8.8% and 4.4% (P < 0.0001) and > 10 mmol/l was 28.9 ± 16.5%, 40.6 ± 12.1% and 55.6 ± 17.9% (P < 0.0001) in the three groups respectively. CONCLUSIONS: Participants achieving HBA1c < 58 mmol/mol (< 7.5%) had ~ 60% of CBG readings in range (4-10 mmol/l), with up to 30% of readings > 10 mmol/l. This target of achieving 60% or more readings within target, and being permissive with up to 30% readings > 10 mmol/l may be a novel target for people with diabetes, and may reduce anxiety associated with readings out of range.

Anatomy and immunochemical characterization of the non-arterial peptidergic diffuse dural innervation of the rat and Rhesus monkey: Implications for functional regulation and treatment in migraine.

Objective The interplay between neuronal innervation and other cell types underlies the physiological functions of the dura mater and contributes to pathophysiological conditions such as migraine. We characterized the extensive, but understudied, non-arterial diffuse dural innervation (DDI) of the rat and Rhesus monkey. Methods We used a comprehensive integrated multi-molecular immunofluorescence labeling strategy to extensively profile the rat DDI and to a lesser extent that of the Rhesus monkey. Results The DDI was distributed across a dense, pervasive capillary network and included free nerve endings of peptidergic CGRP-expressing C fibers that were closely intertwined with noradrenergic (NA) sympathetic fibers and thin-caliber nonpeptidergic "C/Aδ" fibers. These newly identified C/Aδ fibers were unmyelinated, like C fibers, but expressed NF200, usually indicative of Aδ fibers, and uniquely co-labeled for the CGRP co-receptor, RAMP1. Slightly-larger caliber NF200-positive fibers co-labeled for myelin basic protein (MBP) and terminated as unbranched corpuscular endings. The DDI peptidergic fibers co-labeled for the lectin IB4 and expressed presumably excitatory α1-adrenergic receptors, as well as inhibitory 5HT1D receptors and the delta opioid receptor (δOR), but rarely the mu opioid receptor (µOR). Labeling for P2X3, TRPV1, TRPA1, and parasympathetic markers was not observed in the DDI. Interpretation These results suggest potential functional interactions, wherein peptidergic DDI fibers may be activated by stress-related sympathetic activity, resulting in CGRP release that could be detected in the circulation. CGRP may also activate nonpeptidergic C/Aδ fibers that are likely mechanosensitive or polymodal, leading to activation of post-synaptic pain transmission circuits. The distribution of α1-adrenergic receptors, RAMP1, and the unique expression of the δOR on CGRP-expressing DDI fibers suggest strategies for functional modulation and application to therapy.

MicroRNA-122 can be measured in capillary blood which facilitates point-of-care testing for drug-induced liver injury.

AIMS: Liver-enriched microRNA-122 (miR-122) is a novel circulating biomarker for drug-induced liver injury (DILI). To date, miR-122 has been measured in serum or plasma venous samples. If miR-122 could be measured in capillary blood obtained from a finger prick it would facilitate point-of-care testing, such as in resource-limited settings that have a high burden of DILI. METHODS: In this study, in healthy subjects, miR-122 was measured by polymerase chain reaction in three capillary blood drops taken from different fingers and in venous blood and plasma (n = 20). miR-122 was also measured in capillary blood obtained from patients with DILI (n = 8). RESULTS: Circulating miR-122 could be readily measured in a capillary blood drop in healthy volunteers with a median (interquartile range) cycle threshold (Ct) of 32.6 (31.1-34.2). The coefficient of variation for intraindividual variability across replicate blood drops was 49.9%. Capillary miR-122 faithfully reflected the concentration in venous blood and plasma (Pearson R = 0.89, P < 0.0001; 0.88, P < 0.0001, respectively). miR-122 was 86-fold higher in DILI patients [median value 1.0 × 108 (interquartile range 1.89 × 107 -3.04 × 109 ) copies/blood drop] compared to healthy subjects [1.85 × 106 (4.92 × 105 -5.88 × 106 ) copies/blood drop]. Receiver operator characteristic analysis demonstrated that capillary miR-122 sensitively and specifically reported DILI (area under the curve: 0.96, P = 0.0002). CONCLUSION: This work supports the potential use of miR-122 as biomarker of human DILI when measured in a capillary blood drop. With development across DILI aetiologies, this could be used by novel point-of-care technologies to produce a minimally invasive, near-patient, diagnostic test.

Differentiating Intralymphatic Histiocytosis, Intravascular Histiocytosis, and Subtypes of Reactive Angioendotheliomatosis: Review of Clinical and Histologic Features of All Cases Reported to Date.

Reactive angioendotheliomatosis (REA) is a rare benign angioproliferative condition of the skin, which has been noted to occur in patients with a variety of underlying systemic diseases. Histopathologically, this condition is characterized by vascular proliferation, and endothelial cell hyperplasia within the lumina and around dermal vessels, without significant cellular atypia. Since the first case of RAE was reported in 1958, multiple histologic patterns of benign cutaneous vascular proliferations with similar clinical presentations to RAE have been described in the literature and have been proposed as subtypes of the originally described condition. Among these entities are diffuse dermal angiomatosis (DDA), acroangiodermatitis, glomeruloid angioendotheliomatosis, and angiomatosis associated with cryoproteins. It has also been proposed that another entity, characterized by the benign proliferation of histiocytes within the lumina of cutaneous vessels, is a subtype of RAE. Histiocytosis within dermal vessels, in conjunction with skin pathology, was first reported in 1994. Based on the appearance of involved vessels, it was initially believed that the histiocytic proliferations were within the lumina of capillaries. Hence, the term intravascular histiocytosis was introduced to describe this histologic finding. However, subsequent introduction of an immunohistochemical (IHC) marker specific for lymphatic vessels demonstrated that most cases of cutaneous histiocyte proliferation are intralymphatic, rather than truly intravascular. However, there have also been reports of IHC-confirmed cases of true intravascular (intracapillary) histiocytosis. In this study, clinical and histologic data from all of the cases of RAE and IHC-confirmed cases of intravascular histiocytosis and intralymphatic histiocytosis reported in the literature to date are examined. Through comparison of the frequency with which key clinical and histologic features present in cases of each group, the authors provide improved clarity of the similarities and differences between these 3 entities.

Capillary and Venous Lactate Agreement: a pilot prospective observational study.

BACKGROUND: Blood lactate is a marker of patient illness severity. Capillary lactate (CAP-LACT) measurement can potentially improve patient screening; however, it has poor evidence of clinical utility. AIM: We aimed to investigate agreement between CAP-LACT and peripheral venous lactate (PV-LACT). METHODS: We performed a prospective observational pilot study of 99 patients requiring lactate measurement. Paired CAP-LACT and PV-LACT was recorded. Agreement was determined by Bland-Altman analysis. RESULTS: Bias was 0.2 mmol/L, with 95% limits of agreement from -1.9 to 2.3. CONCLUSIONS: CAP-LACT has poor agreement with PV-LACT. Further research is needed to improve its potential clinical utility.

Utilization of HPASubC for the Identification of Sinusoid-Specific Proteins in the Liver.

Mass spectrometry-based proteomes of human organs and tissues are powerful tools but fail to capture protein localization and expression at the cellular level. For example, the proteome signal in liver represents the combined protein expression across diverse cellular constituents that include hepatocytes, Kupffer cells, endothelial cells, and others. We utilized HPASubC and the Human Protein Atlas (HPA) to identify the sinusoidal component of protein liver expression to further subset and organize this homogeneous signal. We evaluated 51 109 liver images covering 13 197 proteins from the HPA and discovered 1054 proteins that were exclusive to sinusoidal cells. Sinusoidal staining patterns were identified in a Kupffer cell (n = 247), endothelial cell (n = 358), or lymphocyte (n = 86) specific pattern. Two-hundred and thirty-nine of these proteins were not present in the NextProt or Human Proteome Map liver data sets, potentially expanding our knowledge of the liver proteome. We additionally demonstrate unique endothelial cell expression patterns that distinguish between portal vein, hepatic artery, capillary sinusoids, and central vein regions. These findings significantly improve our understanding of the liver proteome with insight into the endothelial complexity across the hepatic vascular network.

Relation of peritubular capillary features to class of lupus nephritis.

BACKGROUND: Experimental studies have linked peritubular capillary (PTC) loss with progression of chronic kidney disease. Minimal information on PTC in lupus nephritis (LN) has been reported. We therefore evaluated the PTC area in different classes of LN and determined if specific clinical characteristics correlated with PTC changes. METHODS: Renal biopsies of 253 subjects with LN (categorized using the ISN/RPS 2003 classification) and 13 normal renal donors (the controls) were retrospectively evaluated for PTC morphology by staining for CD31 with immunohistochemistry method. The percent positive area of PTC (% PTC) was correlated with serum and urinary measures of renal function and renal pathology. RESULTS: Significant PTC loss was observed in all classes of LN compared to controls. The % PTC area was highest in controls (7.64±1.48 %) with levels of 1.95±1.50, 4.16±3.85, 4.19±4.45, 5.02±1.79, and 4.45±3.75 in classes II, III, IV, IV combined with V and V, respectively (all p values < 0.05). The lowest PTC density was observed in class II LN, but this may be because some cases with worse classes of LN showed increased PTC density due to abnormally dilated capillaries associated with acute inflammation and angiogenesis. %PTC was increased in those with hematuria (5.8±5.2 vs. 3.6±3.4 %, red blood cells 3-10 vs. < 3 cells/high power field, p < 0.05) and was reduced in those with a moderately declined renal function (3.29±3.40 vs. 4.42±4.12, eGFR 15-59 vs. ≥ 60 ml/min/1.73 m2, p < 0.05). Nephrotic-range proteinuria also trended to be associated with lower PTC density although it did not reach statistical significance (3.1±2.6 vs. 4.9±4.5, p= 0.067). CONCLUSIONS: LN is associated with PTC loss and the severity correlates with reduced renal function. Further studies are needed to investigate whether a loss of PTC can predict long term renal outcomes in LN.

Abnormal Morphology of Blood Vessels in Erythematous Skin From Atopic Dermatitis Patients.

Previous studies suggest that altered peripheral blood circulation might be associated with erythema or inflammation in atopic dermatitis (AD) patients. However, the overall structure of blood vessels and capillaries in AD skin is poorly understood because most studies have involved light-microscopic observation of thin skin sections. In the present study, we compared the 3-dimensional structures of peripheral blood vessels of healthy subjects and AD patients in detail by means of 2-photon microscopy. In skin from healthy subjects, superficial vascular plexus and capillaries originating from flexous blood vessels were observed. However, skin from AD patients contained thickened, flexuous blood vessels, which might be associated with increased blood flow, in both erythematous and nonlesional areas. However, patients with lichenification did not display these morphological changes. Bifurcation of vessels was not observed in either erythematous or lichenification lesions. These results might be helpful for developing new clinical strategies to treat erythema in AD patients.

Complement Factor C4d Is a Common Denominator in Thrombotic Microangiopathy.

Complement activation has a major role in thrombotic microangiopathy (TMA), a disorder that can occur in a variety of clinical conditions. Promising results of recent trials with terminal complement-inhibiting drugs call for biomarkers identifying patients who might benefit from this treatment. The primary aim of this study was to determine the prevalence and localization of complement factor C4d in kidneys of patients with TMA. The secondary aims were to determine which complement pathways lead to C4d deposition and to determine whether complement activation results in deposition of the terminal complement complex. We examined 42 renal sections with histologically confirmed TMA obtained from a heterogeneous patient group. Deposits of C4d, mannose-binding lectin, C1q, IgM, and C5b-9 were scored in the glomeruli, peritubular capillaries, and arterioles. Notably, C4d deposits were present in 88.1% of TMA cases, and the various clinical conditions had distinct staining patterns within the various compartments of the renal vasculature. Classical pathway activation was observed in 90.5% of TMA cases. C5b-9 deposits were present in 78.6% of TMA cases and in 39.6% of controls (n=53), but the staining pattern differed between cases and controls. In conclusion, C4d is a common finding in TMA, regardless of the underlying clinical condition. Moreover, C5b-9 was present in >75% of the TMA samples, suggesting that terminal complement inhibitors may have a beneficial effect in these patients. C4d and C5b-9 should be investigated as possible diagnostic biomarkers in the clinical work-up of patients suspected of having complement-mediated TMA.

[Pathology and differential diagnosis of immunotactoid glomerulopathy].

Objective: To study the morphologic changes of immunotactoid glomerulopathy and to investigate the clinical pathological features and differential diagnosis. Methods: Renal biopsy was observed under the light microscope, immunofluorescence and electron microscopy in a case of newly diagnosed immunotactoid glomerulopathy. Results: This patient clinically presented with nephrotic syndrome and hypertension, without family history of renal diseases. Light microscopy showed that diffusely massive and specific protein deposition in the glomerulus in Masson staining. Immunofluorescence revealed IgG, C3 and κ were deposited along the capillary walls and mesangial regions. Electron microscopic examination showed that a large amount of microtubule like substances and a small amount of long bar-shaped and dense crystal-like substances were deposited in the subendothelial spaces and mesangial areas. Conclusions: Light microscopy and immunofluorescence of immunotactoid glomerulopathy show no specifically pathological changes. Under electron microscope, a large amount of microtubule like substances is deposited in the glomerulus, which is the key point to distinguish this disease from other glomerular diseases. Except for the microtubule-like substances, the present case is accompanied by the deposition of long bar-shaped and dense crystal-like substance, which has not been reported in previous studies.

Correlation of disease activity in proliferative glomerulonephritis with glomerular spleen tyrosine kinase expression.

Spleen tyrosine kinase (SYK) is an important component of the intracellular signaling pathway for various immunoreceptors. Inhibition of SYK has shown promise in preclinical models of autoimmune and glomerular disease. However, the description of SYK expression in human renal tissue, which would be desirable ahead of clinical studies, is lacking. Here we conducted immunohistochemical analysis for total and phosphorylated SYK in biopsy specimens from >120 patients with a spectrum of renal pathologies, including thin basement membrane lesion, minimal change disease, membranous nephropathy, IgA nephropathy, lupus nephritis, ANCA-associated glomerulonephritis, antiglomerular basement membrane disease, and acute tubular necrosis. We found significant SYK expression in proliferative glomerulonephritis and that glomerular expression levels correlated with presenting serum creatinine and histological features of disease activity that predict outcome in IgA nephropathy, lupus nephritis, ANCA-associated glomerulonephritis, and antiglomerular basement membrane disease. SYK was phosphorylated within pathological lesions, such as areas of extracapillary and endocapillary proliferation, and appeared to localize to both infiltrating leucocytes and to resident renal cells within diseased glomeruli. Thus SYK is associated with the pathogenesis of proliferative glomerulonephritides, suggesting that these conditions may respond to SYK inhibitor treatment.

An immunohistochemical analysis of SERT in the blood-brain barrier of the male rat brain.

5-Hydroxytryptamine (5-HT) was originally discovered as a vasoconstrictor. 5-HT lowers blood pressure when administered peripherally to both normotensive and hypertensive male rats. Because the serotonin transporter (SERT) can function bidirectionally, we must consider whether 5-HT can be transported from the bloodstream to the central nervous system (CNS) in facilitating the fall in blood pressure. The blood-brain barrier (BBB) is a highly selective barrier that restricts movement of substances from the bloodstream to the CNS and vice versa, but the rat BBB has not been investigated in terms of SERT expression. This requires us to determine whether the BBB of the rat, the species in which we first observed a fall in blood pressure to infused 5-HT, expresses SERT. We hypothesized that SERT is present in the BBB of the male rat. To test this hypothesis, over 500 blood vessels were sampled from coronal slices of six male rat brains. Immunofluorescence of these coronal slices was used to determine whether SERT and RecA-1 (an endothelial cell marker) colocalized to the BBB. Blood vessels were considered to be capillaries if they were between 1.5 and 23 µm (intraluminal diameter). SERT was identified in the largest pial vessels of the BBB (mean ± SEM = 228.70 ± 18.71 µm, N = 9) and the smallest capillaries (mean ± SEM = 2.75 ± 0.12 µm, N = 369). SERT was not identified in the endothelium of blood vessels ranging from 20 to 135 µm (N = 45). The expression of SERT in the rat BBB means that 5-HT entry into the CNS must be considered a potential mechanism when investigating 5-HT-induced fall in blood pressure.

Intraoperative fluorescence-based enhanced reality laparoscopic real-time imaging to assess bowel perfusion at the anastomotic site in an experimental model.

BACKGROUND: Fluorescence videography is a promising technique for assessing bowel perfusion. Fluorescence-based enhanced reality (FLER) is a novel concept, in which a dynamic perfusion cartogram, generated by computer analysis, is superimposed on to real-time laparoscopic images. The aim of this experimental study was to assess the accuracy of FLER in detecting differences in perfusion in a small bowel resection-anastomosis model. METHODS: A small bowel ischaemic segment was created laparoscopically in 13 pigs. Animals were allocated to having anastomoses performed at either low perfusion (25 per cent; n = 7) or high perfusion (75 per cent; n = 6), as determined by FLER analysis. Capillary lactate levels were measured in blood samples obtained by serosal puncturing in the ischaemic area, resection lines and vascularized areas. Pathological inflammation scoring of the anastomosis was carried out. RESULTS: Lactate levels in the ischaemic area (mean(s.d.) 5·6(2·8) mmol/l) were higher than those in resection lines at 25 per cent perfusion (3·7(1·7) mmol/l; P = 0·010) and 75 per cent perfusion (2·9(1·3) mmol/l; P < 0·001), and higher than levels in vascular zones (2·5(1·0) mmol/l; P < 0·001). Lactate levels in resection lines with 75 per cent perfusion were lower than those in lines with 25 per cent perfusion (P < 0·001), and similar to those in vascular zones (P = 0·188). Levels at resection lines with 25 per cent perfusion were higher than those in vascular zones (P = 0·001). Mean(s.d.) global inflammation scores were higher in the 25 per cent perfusion group compared with the 75 per cent perfusion group for mucosa/submucosa (2·1(0·4) versus 1·2(0·4); P = 0·003) and serosa (1·8(0·4) versus 0·8(0·8); P = 0·014). A ratio of preanastomotic lactate levels in the ischaemic area relative to the resection lines of 2 or less was predictive of a more severe inflammation score. CONCLUSION: In an experimental model, FLER appeared accurate in discriminating bowel perfusion levels. Surgical relevance Clinical assessment has limited accuracy in evaluating bowel perfusion before anastomosis. Fluorescence videography estimates intestinal perfusion based on the fluorescence intensity of injected fluorophores, which is proportional to bowel vascularization. However, evaluation of fluorescence intensity remains a static and subjective measure. Fluorescence-based enhanced reality (FLER) is a dynamic fluorescence videography technique integrating near-infrared endoscopy and specific software. The software generates a virtual perfusion cartogram based on time to peak fluorescence, which can be superimposed on to real-time laparoscopic images. This experimental study demonstrates the accuracy of FLER in detecting differences in bowel perfusion in a survival model of laparoscopic small bowel resection-anastomosis, based on biochemical and histopathological data. It is concluded that real-time imaging of bowel perfusion is easy to use and accurate, and should be translated into clinical use.