Latent autoimmune diabetes of adulthood (LADA) is associated with small fibre neuropathy.

AIM: To assess if latent autoimmune diabetes of adulthood (LADA) is associated with small fibre neuropathy. METHODS: Participants with LADA (n=31), Type 2 diabetes (n=31) and healthy control participants without diabetes (n=31) underwent a detailed assessment of neurologic deficits, quantitative sensory testing, electrophysiology, skin biopsy and corneal confocal microscopy. RESULTS: The groups were matched for age (healthy control without diabetes: 53.5±9.1 vs. Type 2 diabetes: 58.0±6.5 vs. LADA: 53.2±11.6 years), duration of diabetes (Type 2 diabetes: 10.0±8.3 vs. LADA: 11.0±9.1 years) and blood pressure. However, BMI (P=0.01) and triglycerides (P=0.0008) were lower and HbA1c (P=0.0005), total cholesterol (P=0.01) and HDL (P=0.002) were higher in participants with LADA compared with Type 2 diabetes. Peroneal motor nerve conduction velocity (P=0.04) and sural sensory nerve conduction velocity (P=0.008) were lower in participants with latent autoimmune diabetes in adults compared with Type 2 diabetes. Intra-epidermal nerve fibre density (P=0.008), corneal nerve fibre density (P=0.003) and corneal nerve branch density (P=0.006) were significantly lower in participants with LADA compared with Type 2 diabetes. There were no significant differences in the other neuropathy parameters. CONCLUSIONS: Despite comparable age and duration of diabetes, participants with LADA demonstrate more severe neuropathy and particularly small fibre neuropathy, compared with participants with Type 2 diabetes.

Distal lower limb strength is reduced in subjects with impaired glucose tolerance and is related to elevated intramuscular fat level and vitamin D deficiency.

AIM: To quantify muscle strength and size in subjects with impaired glucose tolerance (IGT) in relation to intramuscular non-contractile tissue, the severity of neuropathy and vitamin D level. METHODS: A total of 20 subjects with impaired glucose tolerance and 20 control subjects underwent assessment of strength and size of knee extensor, flexor and ankle plantar and dorsi-flexor muscles, as well as quantification of intramuscular non-contractile tissue and detailed assessment of neuropathy and serum 25-hydroxy vitamin D levels. RESULTS: In subjects with impaired glucose tolerance, proximal knee extensor strength (P = 0.17) and volume (P = 0.77), and knee flexor volume (P = 0.97) did not differ from those in control subjects. Ankle plantar flexor strength was significantly lower (P = 0.04) in the subjects with impaired glucose tolerance, with no difference in ankle plantar flexor (P = 0.62) or dorsiflexor volume (P = 0.06) between groups. Intramuscular non-contractile tissue level was significantly higher in the ankle plantar flexors and dorsiflexors (P = 0.03) of subjects with impaired glucose tolerance compared with control subjects, and it correlated with the severity of neuropathy. Ankle plantar flexor muscle strength correlated significantly with corneal nerve fibre density (r = 0.53; P = 0.01), a sensitive measure of small fibre neuropathy, and was significantly lower in subjects with vitamin D deficiency (P = 0.02). CONCLUSIONS: People with impaired glucose tolerance have a significant reduction in distal but not proximal leg muscle strength, which is not associated with muscle atrophy, but with increased distal intramuscular non-contractile tissue, small fibre neuropathy and vitamin D deficiency.

Altered walking strategy and increased unsteadiness in participants with impaired glucose tolerance and Type 2 diabetes relates to small-fibre neuropathy but not vitamin D deficiency.

AIMS: To investigate alterations in walking strategy and dynamic sway (unsteadiness) in people with impaired glucose tolerance and people with Type 2 diabetes in relation to severity of neuropathy and vitamin D levels. METHODS: A total of 20 people with Type 2 diabetes, 20 people with impaired glucose tolerance and 20 people without either Type 2 diabetes or impaired glucose tolerance (control group) underwent gait analysis using a motion analysis system and force platforms, and detailed assessment of neuropathy and serum 25 hydroxy-vitamin D levels. RESULTS: Ankle strength (P = 0.01) and power (P = 0.003) during walking and walking speed (P = 0.008) were preserved in participants with impaired glucose tolerance but significantly lower in participants with Type 2 diabetes compared with control participants; however, step width (P = 0.005) and dynamic medio-lateral sway (P = 0.007) were significantly higher and posterior maximal movement (P = 0.000) was lower in participants with impaired glucose tolerance, but preserved in those with Type 2 diabetes compared with the control group. Dynamic medio-lateral sway correlated with corneal nerve fibre length (P = 0.001) and corneal nerve branch density (P = 0.001), but not with vibration perception threshold (P = 0.19). Serum 25 hydroxy-vitamin D levels did not differ significantly among the groups (P = 0.10) and did not correlate with any walking variables or measures of dynamic sway. CONCLUSIONS: Early abnormalities in walking strategy and dynamic sway were evident in participants with impaired glucose tolerance, whilst there was a reduction in ankle strength, power and walking speed in participants with Type 2 diabetes. Unsteadiness correlated with small-, but not large-fibre neuropathy and there was no relationship between vitamin D levels and walking variables.

Acquisition of biologically relevant gene expression data by Affymetrix microarray analysis of archival formalin-fixed paraffin-embedded tumours.

Robust protocols for microarray gene expression profiling of archival formalin-fixed paraffin-embedded tissue (FFPET) are needed to facilitate research when availability of fresh-frozen tissue is limited. Recent reports attest to the feasibility of this approach, but the clinical value of these data is poorly understood. We employed state-of-the-art RNA extraction and Affymetrix microarray technology to examine 34 archival FFPET primary extremity soft tissue sarcomas. Nineteen arrays met stringent QC criteria and were used to model prognostic signatures for metastatic recurrence. Arrays from two paired frozen and FFPET samples were compared: although FFPET sensitivity was low ( approximately 50%), high specificity (95%) and positive predictive value (92%) suggest that transcript detection is reliable. Good agreement between arrays and real time (RT)-PCR was confirmed, especially for abundant transcripts, and RT-PCR validated the regulation pattern for 19 of 24 candidate genes (overall R(2)=0.4662). RT-PCR and immunohistochemistry on independent cases validated prognostic significance for several genes including RECQL4, FRRS1, CFH and MET - whose combined expression carried greater prognostic value than tumour grade - and cmet and TRKB proteins. These molecules warrant further evaluation in larger series. Reliable clinically relevant data can be obtained from archival FFPET, but protocol amendments are needed to improve the sensitivity and broad application of this approach.

Neovascularization in early atherosclerotic lesions of human carotid arteries: its potential contribution to plaque development.

Neovascularization is a prominent feature of late-stage atherosclerotic lesions and their complications but is generally regarded as an insignificant, undetectable component of the earliest stages of plaque development, probably because of relatively poor histological techniques. Using an improved vascular staining procedure, we have examined the extent of neovascularization in the earliest plaque lesions. Combined monoclonal antibodies to CD31, CD34, and von Willebrand factor have provided an ultrasensitive technique with which to visualize blood vessels in early atherosclerotic lesions (n = 55) of human carotid arteries obtained through surgical procedures. Capillary-like microvessels were shown in very early atherosclerotic lesions (type II), where they were associated with the distribution of macrophages and a few immature mast cells. Neovascularization was more prominent in type III lesions with vessels of variable size, often providing a focus around which local accumulations of macrophages and apolipoproteins A-I and B were visualized. Thickened type III lesions usually showed an intricate network of microvessels, together with numerous mast cells. These studies have shown neovascularization as a prominent feature of early stages of atherosclerotic plaque development. Whereas distribution of apolipoproteins A-I and B were observed in the very earliest stages of the plaque intima, these lipids, together with macrophages, foam cells, and mast cells, were observed as perivascular accumulations in a proportion of type II and III lesions. Such findings indicate that neovascularization is an important feature of early plaque development and may provide an additional or alternative source of leukocyte and lipid accumulations relative to the arterial lumen.

Evidence for in vivo mitosis by granule-containing mast cells from canine mastocytomas.

Mast cell accumulations are generally considered to arise almost exclusively from the recruitment of non-granulated, bone-marrow-derived, precursor cells, with the stem cell factor (SCF) reported to play a crucial role in the growth, development and maturation of granulated mast cells within specific tissue sites. In this study dog mastocytoma specimens have been examined by both immunohistochemical and ultrastructural techniques, to demonstrate that fully granulated mast cells are capable of mitotic activity. Observations showing the formation of mitotic spindles, chromosome separation and cytokinesis all support the concept that granulated mast cells are capable of proliferative activity. The ability of mature granulated mast cells to replicate provides an alternative process for local increases in mast cell numbers, at least in canine mast cell tumours. Such observations suggest the possibility that normal or neoplastic human mast cells, fully granulated, have the potential to proliferate in specific tissue sites.

Observations on bone formation and remodelling in advanced atherosclerotic lesions of human carotid arteries.

Immunolocalisation and histochemical techniques were used to examine mineralised bone deposits within late stage atherosclerotic plaques of human carotid arteries. These specimens showed characteristic features of osteogenesis. Large calcifications were often observed in close association with or integrated within mineralised bone. Smooth muscle cells (alpha-actin positive) were often located around osteoid-like matrix, together with focal accumulations of macrophages (CD68 and HAM56 positive). Local accumulations of mast cells (tryptase positive) were consistently observed in close association with the bone. Multinucleated giant cells in close apposition with mineralised bone demonstrated typical osteoclastic morphology, and were positively stained for acid phosphatase and the macrophage marker CD68. Thus, all the normal features of bone formation and resorption were observed in this microcosm of osteogenesis within atherosclerotic plaque; the term 'osteosome' seems appropriate for the structure. These osteosomes have numerous advantages for experimental studies of the various osteogenic factors responsible for bone metabolism, especially following short-term tissue culture. This ex vivo technique was used to demonstrate the distribution and the multiple cellular sources of bone morphogenetic protein 6.

Clinical, biochemical, and immunohistochemical features of necrobiotic xanthogranulomatosis.

AIMS: To describe the clinical features of two patients with paraproteinaemia and necrobiotic xanthogranulomatosis together with detailed immunohistochemistry of the lesions in one. METHODS: The clinical history and results of biochemical investigations of the patients were retrieved from the files. Immunohistochemistry was used to investigate the expression of macrophage and mast cell markers, amyloid A and P, S-100 protein, and apolipoprotein AI and B in xanthogranulomatous skin lesions from patient 2. In addition, protein A-sepharose chromatography was used to separate serum from patient 2 and apolipoprotein B and the IgG paraprotein were measured in the fractions eluted. RESULTS: Monocytes/macrophages comprised the major cellular component of the lesion, and unusually for xanthomata, areas of collagen necrosis were also seen. Activated mast cells were present at the margins of macrophage clusters and adjacent to areas of collagen necrosis. Serum paraprotein was bound to low density lipoproteins as judged by protein A-sepharose chromatography, and was also located within macrophagic foam cells of the lesion on immunohistochemistry. CONCLUSIONS: These observations demonstrate many features similar to atherosclerosis including collagen necrosis and mast cell activation.

Evidence against a significant role for mast cells in blastocyst implantation in the rat and mouse.

Rats were treated with the highly potent stabilizer of mast cells, FPL 55618, before and during the first seven days of pregnancy to establish whether stabilization of mast cells resulted in impaired blastocyst implantation. There was no significant reduction in either the number of ovulations or the number of implantation sites in treated rats compared with controls; 11 of 15 treated rats were pregnant compared with 5 of 6 control rats. The distribution of mast cells was examined in uterine tissues, implantation sites and interimplantation sites in both rats and mice using highly sensitive immunohistochemical techniques. Virtually all of the mast cells in rat uterine tissue stained for rat mast cell protease-I (RMCP-I; connective tissue type), whereas few stained for RMCP-II (mucosal type). Most of the mast cells were present in the myometrium with very sparse distribution in the endometrium and there were no differences in numbers of mast cells between implantation and inter-implantation sites on Day 7 of pregnancy. In tissue sections of mouse uteri sampled from Day 1 to Day 8 of pregnancy there were virtually no mast cells in the endometrium or deciduum adjacent to implantation sites. Mouse uterine mast cells also stained predominantly for the connective tissue-type mast cell protease MMCP-4, the murine equivalent of RMCP-I. Thus, mast cells and their products appear to play little, if any, role in blastocyst implantation in murid rodents. Since mast cells are a prominent feature of human endometrium, this study emphasizes the important consideration of species differences when choosing animal models for implantation studies.

The results of topical application of 13-cis-retinoic acid on basal cell carcinoma. A correlation of the clinical effect with histopathological examination and serum retinol level.

A group of 50 patients with basal cell carcinoma of the face was treated by 13-cis-retinoic acid. The treatment resulted in diminution of the tumors. Complete regression was observed in 4 cases. Histological examination revealed necrosis of cancer cells and mononuclear infiltration into the treated tumors. In the group with weak clinical and histological reaction to the treatment all basal cell carcinomas were of adenoid type. A better effect was observed in the group with lower serum retinol level. This treatment method seems to be supplementary to surgery in prevention of the tumor recurrence.

Mast cells in cervical ripening--an immunohistochemical and biomechanical study in rats.

Cervical ripening purportedly involves different cell types and mediators normally associated with inflammatory reactions. The purpose of the present study was to determine the presence of mast cells in rat cervices during spontaneous and antigestagen induced ripening and to test whether a mast cell stabilizer was able to inhibit the antigestagen induced cervical ripening. Immunohistochemical examinations demonstrated an increased number of mast cells in pregnant and intrapartum rats. Furthermore, mast cell degranulation was found to be prominent after antigestagen treatment. The degranulation was completely abolished by co-treatment with the mast cell stabilizer. Biomechanical analysis showed that the mast cell stabilizer also inhibited the antigestagen induced cervical ripening to some extent. Thus, it is concluded that mast cell stabilizers might constitute a new approach in the treatment of preterm cervical ripening.

Small fiber neuropathy in diabetes: clinical consequence and assessment.

Recent findings have shed new light on the role of peripheral nerves in the skin and have established a modern concept of cutaneous neurobiology. There is bidirectional rather than unidirectional (conveying information from the periphery) signaling between central and peripheral nerves and the endocrine and immune systems. This interaction is mediated principally by cutaneous small nerve fibers and will influence a variety of physiologic and pathophysiologic functions central to wound healing, which include cellular development, growth, differentiation, immunity, vasoregulation, and leukocyte recruitment. Thus, disease of the small fibers in diabetic patients is frequent and may have a considerable impact on the predisposition and subsequent wound-healing response to foot ulceration. The authors review the basic pathophysiology, clinical consequences, and current methods to evaluate somatic and autonomic nerve fiber dysfunction and damage.

The Neuropad test: a visual indicator test for human diabetic neuropathy.

AIMS/HYPOTHESIS: The commercially available Neuropad test was developed as a simple visual indicator test to evaluate diabetic neuropathy. It uses a colour change to define the integrity of skin sympathetic cholinergic innervation. We compared the results of Neuropad assessment in the foot with established measures of somatic and autonomic neuropathy. METHODS: Fifty-seven diabetic patients underwent Neuropad assessment, quantitative sensory and autonomic function testing, and evaluation of intra-epidermal nerve fibre density in foot skin biopsies. RESULTS: Neuropad responses correlated with the neuropathy disability score (r(s)=0.450, p<0.001), neuropathic symptom score (r(s)=0.288, p=0.03), cold detection threshold (r(s)=0.394, p = 0.003), heat-as-pain perception threshold visual analogue score 0.5 (r(s)=0.279, p=0.043) and deep-breathing heart rate variability (r(s)= -0.525, p<0.001). Intra-epidermal nerve fibre density (fibres/mm) compared with age- and sex-matched control subjects (11.06+/-0.82) was non-significantly reduced (7.37+/-0.93) in diabetic patients with a normal Neuropad response and significantly reduced in patients with a patchy (5.01+/-0.93) or absent (5.02+/-0.77) response (p=0.02). The sensitivity of an abnormal Neuropad response in detecting clinical neuropathy (neuropathy disability score >or=5) was 85% (negative predictive value 71%) and the specificity was 45% (positive predictive value 69%). CONCLUSIONS/INTERPRETATION: The Neuropad test may be a simple indicator for screening patients with diabetic neuropathy.

Contribution of VCAF-positive cells to neovascularization and calcification in atherosclerotic plaque development.

Calcification of the vessel wall is a regulated process with many similarities to osteogenesis. Progenitor cells may play a role in this process. Previously, we identified a novel gene, Vascular Calcification Associated Factor (VCAF), which was shown to be important in pericyte osteogenic differentiation. The aim of this study was to determine the localization and expression pattern of VCAF in human cells and tissues. Immunohistochemical analysis of seven atherosclerotic arteries confirmed VCAF protein expression within calcified lesions. In addition, individual VCAF-positive cells were detected within the intima and adventitia in areas where sporadic 3G5-positive pericytes were localized. Furthermore, VCAF-positive cells were identified in newly formed microvessels in association with CD34-positive/CD146-positive/c-kit-positive cells as well as in intact CD31-positive endothelium in internal mammary arteries. Western blot analysis confirmed the presence of VCAF (18 kD) in protein lysates extracted from human smooth muscle cells, endothelial cells, macrophages, and osteoblasts. In fracture callus samples from three patients, VCAF was detected in osteoblasts and microvessels. This study demonstrates the presence of VCAF in neovessels and raises the possibility that VCAF could be a new marker for vascular progenitor cells involved in a number of differentiation pathways. These data may have implications for the prevention or treatment of vascular disease.

Lymphatic vessel density, microvessel density and lymphangiogenic growth factor expression in colorectal cancer.

OBJECTIVE: Microvessel density (MVD) has been studied as a prognostic marker in human cancers. Quantification of lymphatic vessel density (LVD) is now possible by using new antibodies. Expression of the lymphangiogenic growth factors, VEGF-C and VEGF-D, is associated with poorer clinicopathological outcomes in various tumours. The aim of this study was to quantify LVD and MVD in colorectal cancer, determine the relationship between LVD, MVD and clinicopathological variables and examine the relationship between LVD and tumour expression of VEGF-C and VEGF-D. METHOD: Thirty primary colorectal cancers were immunostained for CD34, lymph vessel endothelial hyaluronan receptor-1 (LYVE-1), VEGF-A and VEGF-D using standard techniques. LVD and MVD were determined by Chalkley grid counting. Tumours were assessed for the presence or absence of LYVE-1 positive lymphatics at different areas within the tumour and the tumour was scored for VEGF-C and VEGF-D immunostaining intensity at the invading tumour edge. Non-parametric tests were used for statistical analysis and a P-value of <0.05 was taken as significant. RESULTS: Lymph vessel endothelial hyaluronan receptor-1 was an excellent lymphatic vessel marker. Within normal bowel wall, lymphatic vessels were found rarely in the superficial colonic mucosa, but were numerous in the submucosa and muscularis propria. In the majority of tumours, lymphatic vessels were located in the peri-tumoural area, intra-tumoural vessels were sparse and tended to be narrow with closed lumina. At the invading tumour edge, VEGF-C expression was higher (P = 0.028) and VEGF-D expression lower (P = 0.011), in tumours in which lymphatic vessels were present. No significant differences between LVD and any clinicopathological variable or route of metastasis were identified. CONCLUSION: Lymphatic vessel density and MVD can be quantified in colorectal carcinoma using immunohistochemical techniques. The balance between expression of VEGF-C and VEGF-D at the invading tumour edge may enhance lymphatic metastasis, by promoting tumour lymphangiogenesis or by activation of pre-existing lymphatic vessels. No relationship was identified between LVD and clinicopathological variables.

Hepatocyte growth factor and c-Met expression in pericytes: implications for atherosclerotic plaque development.

Intraplaque neovascularization contributes to the progression of atherosclerosis. Our aim is to understand the mobilization of cells and factors involved in this process. We investigated the localization of hepatocyte growth factor (HGF) and its receptor, c-Met, in human atherosclerotic plaques, together with the effects of HGF on pericyte migration in vitro. Atherosclerotic femoral arterial segments were collected and analysed from 13 subjects who were undergoing lower limb amputation. Pericytes were identified in human lesions using a 3G5 antibody. Immunohistochemical analysis localized HGF mainly around microvessels, in association with some, but not all, CD31-positive endothelial cells. c-Met expression was mainly associated with smooth muscle cells and pericytes, around some, but not all, microvessels within the atherosclerotic lesions; no detection was apparent in normal internal mammary arteries. Using RT-PCR, we demonstrated expression of HGF and c-Met in a rat pericyte cell-line, TR-PCT1, and in primary pericytes. HGF treatment of TR-PCT1 cells induced their migration, but not their proliferation, in a dose-dependent manner (10-100 ng/ml, p<0.01), an effect mediated by activation of the serine/threonine kinase Akt, shown by western blot analysis. Treating the cells with the PI3K inhibitors Wortmannin (0.1 microM) or LY294002 (10 microM) abolished these effects. This work demonstrates the expression of c-Met and HGF in human atherosclerotic arteries, in association with SM-actin-positive cells and CD-31-positive cells, respectively. HGF induces pericyte migration via PI3-kinase and Akt activation in vitro. HGF and c-Met may be involved in neovascularization during plaque development, and may recruit pericytes to neovessels. Since pericytes are thought to mechanically stabilize new blood vessels, these factors may function to protect against haemorrhage.

Expression of osteonectin and matrix Gla protein in scleroderma patients with and without calcinosis.

OBJECTIVES: Our aim was to evaluate (i) whether the bone matrix proteins osteonectin and matrix gamma-carboxyglutamic acid protein (MGP) are up-regulated in skin biopsies from patients with systemic sclerosis (SSc) and (ii) whether there is differential expression between patients with and without dermal calcinosis, a distressing and debilitating complication of SSc. METHODS: Skin punch biopsies were taken from the forearms of 38 SSc patients with the limited cutaneous subtype of SSc [17 without calcinosis (lcSSc) and 21 with calcinosis (lcSScCal)] and from 11 healthy control subjects. Immunohistochemistry was performed with antibodies to osteonectin and MGP. Staining was assessed semiquantitatively in the microvascular endothelium and in dermal fibroblasts. The Kruskal-Wallis one-way ANOVA was used to compare the data between patient groups. RESULTS: Both lcSSc and lcSScCal groups showed a statistically significant increase in the percentage of microvessels with osteonectin-positive endothelial cells (EC) (especially the lcSScCal group), whereas lcSScCal alone showed an increase in the percentage of microvessels with MGP-positive EC when compared with controls. In both SSc groups, the percentage of osteonectin and MGP-stained fibroblasts was increased in the reticular dermis (for osteonectin this was more marked in the lcSScCal group). In the papillary dermis, the percentage of osteonectin-stained fibroblasts was increased in both SSc groups but the lcSScCal group alone had a higher percentage of MGP-stained fibroblasts. CONCLUSIONS: When compared with controls, protein expression of osteonectin and MGP was greater in SSc patients generally, and osteonectin expression was significantly higher in EC and fibroblasts of the lcSScCal patients than the lcSSc patients without calcinosis.