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HIV-associated sensory neuropathy (HIV-SN) affects 14–38% of HIV+ individuals stable on therapy with no neurotoxic drugs. Polymorphisms in CAMKK2, P2X7R and P2X4R associated with altered risk of HIV-SN in Indonesian and South African patients. The role of CaMKK2 in neuronal repair makes this an attractive candidate, but a direct role for any protein is predicated on expression in affected tissues. Here, we describe expression of CaMKK2, P2X7R and P2X4R proteins in skin biopsies from the lower legs of HIV+ Indonesians with and without HIV-SN, and healthy controls (HC). HIV-SN was diagnosed using the Brief Peripheral Neuropathy Screen. Biopsies were stained to detect protein gene product 9.5 on nerve fibres and CaMKK2, P2X7R or P2X4R, and were examined using 3-colour sequential scanning confocal microscopy. Intraepidermal nerve fibre densities (IENFD) were lower in HIV+ donors than HC and correlated directly with nadir CD4 T-cell counts (r = 0.69, p = 0.004). However, IENFD counts were similar in HIV-SN+ and HIV-SN− donors (p = 0.19) and so did not define neuropathy. CaMKK2+ cells were located close to dermal and epidermal nerve fibres and were rare in HC and HIV-SN− donors, consistent with a role for the protein in nerve damage and/or repair. P2X7R was expressed by cells in blood vessels of HIV-SN− donors, but rarely in HC or HIV-SN+ donors. P2X4R expression by cells in the epidermal basal layer appeared greatest in HIV-SN+ donors. Overall, the differential expression of CaMKK2, P2X7R and P2X4R supports the genetic evidence of a role for these proteins in HIV-SN.

We report three babies from two families with a severe lethal form of congenital cutis laxa. All three had redundant and doughy-textured skin and two siblings from one family had facial dysmorphism. Echocardiograms showed thickened and poorly contractile hearts, arterial dilatation and tortuosity. Post-mortem examination in two of the babies further revealed widespread ectasia and tortuosity of medium and large sized arteries, myocardial hypertrophy, rib and skull fractures. The presence of fractures initially suggested a diagnosis of osteogenesis imperfecta. Under light microscopy bony matrices were abnormal and arterial wall architecture was grossly abnormal showing fragmented elastic fibres. Molecular analysis of known cutis laxa genes did not yield any pathogenic defects. Whole exome sequencing of DNA following informed consent identified two separate homozygous variants in the LOX (Lysyl Oxidase) gene. LOX belongs to the 5-lysyl oxidase gene family involved in initiation of cross-linking of elastin and collagen. A mouse model of a different variant in this gene recapitulates the phenotype seen in the three babies. Our findings suggest that the LOX gene is a novel cause of severe congenital cutis laxa with arterial tortuosity, bone fragility and respiratory failure.

Response Gene to Complement (RGC)-32 is a cell cycle regulator induced by complement activation, growth factors and cytokines. RGC-32 mediates TGF-β dependent profibrotic pathways, while in immune cells promotes the differentiation of Th17 cells in vitro and in vivo. RGC-32 expression is increased in tubules and glomerular infiltrating cells in kidney biopsies of patients with lupus nephritis. We used the nephrotoxic nephritis (NTN) mouse model to assess whether RGC-32 plays a local role in immune complex mediated glomerulonephritis (GN). NTN was induced in WT and RGC-32−/− mice. Proteinuria, blood urea nitrogen and kidney histopathology were determined to assess kidney function and damage, IgG and C3 deposition. Single cell suspension of kidneys and spleens were analyzed by flow cytometry. Expression of IL-17A, collagen I, III and IV was determined by RT-PCR. RGC-32−/− mice exhibit attenuated renal damage as shown by decreased proteinuria and glomerular scores, as well as decreased frequency of infiltrating PMN and decreased mRNA expression for IL-17A, collagen I, III and IV compared to WT mice. RGC-32 deficiency did not interfere with the induction of NTN as mouse anti-sheep IgG titers, percentage of splenic germinal center B cells, plasma cells, effector CD4+ T cells, Tregs, IL-17A, IFNg secreting cells and kidney deposition of autologous antibodies did not differ between RGC-32−/− and WT mice. These results suggest that RGC-32 contributes to the pathogenesis of immune complex mediated GN by promoting proinflammatory and profibrotic pathways. These data support further efforts to examine the mechanisms by which RGC-32 modulates these pathways and suggest that RGC-32 is a potential novel therapeutic target in the treatment of LN.

Flaviviruses cause febrile illnesses in humans that may progress to encephalitis and death. Both viral and host factors determine the level of virus replication and outcome of infection. In mice, genetically determined resistance conferred by the flavivirus resistance locus (Flv) is responsible for the restricted flavivirus replication and prevention of disease development. Majority of flaviviruses express significant virulence, replicate to high titers and cause high mortality in susceptible mice, while congenic resistant mice endure the infection, show significantly reduced levels of virus replication and remain healthy. In contrast, infection with West Nile virus subtype Kunjin (KUNV) causes morbidity and fatal outcomes even in mice that are naturally resistant to flaviviruses. There are two possible mechanisms that could account for such an unforeseen virulence of KUNV in resistant mice: (a) an abrogation of Flv-controlled natural resistance leading to high virus replication, or (b) massive virus-induced immunopathology in the brain. To identify the cause(s) of fatality of KUNV infection, disease progression, virus replication and brain histopathology were studied in parallel in resistant and congenic susceptible mice. While KUNV replicated to high titers causing early fatalities in susceptible mice, it showed only reduced replication associated with the delayed morbidity in resistant mice indicating no abrogation of the Flv resistance. No evidence of excessive immune cell infiltration and tissue damage following KUNV infection were found. However, incomplete KUNV clearance not previously described was perceived as an important source of pathogenesis in resistant mice.

The representative survey studies provide a comprehensive database on the public awareness and perception of CCS in six selected European countries. Our results provide insights into the public understanding and knowledge of energy related issues and CCS topics. The embedded experimental research provides insights into how information affects CCS perceptions. The results discuss implications for CCS communication methods.

Sir,Epithelial-myoepithelial tumour (EMT) is an uncommon neoplasm composed of myoepithelial cells with varying amounts of duct-forming epithelium. It most frequently occurs in the salivary glands a...

The evidence that Robert Gallo and his colleagues presented on 4th May 1984 regarding HTLV-III (HIV) isolation and the role of HIV in the pathogenesis of AIDS is critically analysed. It is concluded that the evidence does not constitute proof of the isolation of a retrovirus, that the virus is exogenous, or that the virus is causally related to AIDS.

Skeletal muscle α-actin (ACTA1) is the major actin in postnatal skeletal muscle. Mutations of ACTA1 cause mostly fatal congenital myopathies. Cardiac α-actin (ACTC) is the major striated actin in adult heart and fetal skeletal muscle. It is unknown why ACTC and ACTA1 expression switch during development. We investigated whether ACTC can replace ACTA1 in postnatal skeletal muscle. Two ACTC transgenic mouse lines were crossed with Acta1 knockout mice (which all die by 9 d after birth). Offspring resulting from the cross with the high expressing line survive to old age, and their skeletal muscles show no gross pathological features. The mice are not impaired on grip strength, rotarod, or locomotor activity. These findings indicate that ACTC is sufficiently similar to ACTA1 to produce adequate function in postnatal skeletal muscle. This raises the prospect that ACTC reactivation might provide a therapy for ACTA1 diseases. In addition, the mouse model will allow analysis of the precise functional differences between ACTA1 and ACTC.

Recent experimental studies have shown the suitability of silk fibroin scaffold (SFS) and porcine-derived acellular collagen I/III scaffold (ACS) as onlay graft materials for tympanic membrane perforation repair. The aims of this study were to further characterize and evaluate the in vivo biocompatibility of SFS and ACS compared with commonly used materials such as Gelfoam and paper in a rat model. The scaffolds were implanted in subcutaneous (SC) tissue and middle ear (ME) cavity followed by histological and otoscopic evaluation for up to 26 weeks. Our results revealed that SFS and ACS were well tolerated and compatible in rat SC and ME tissues throughout the study. The tissue response adjacent to the implants evaluated by histology and otoscopy showed SFS and ACS to have a milder tissue response with minimal inflammation compared to that of paper. Gelfoam gave similar results to SFS and ACS after SC implantation, but it was found to be associated with pronounced fibrosis and osteoneogenesis after ME implantation. It is concluded that SFS and ACS both were biocompatible and could serve as potential alternative scaffolds for tissue engineering in the ear.

We have examined the histological and ultrastructural features of CNS infection with Murray Valley encephalitis (MVE) virus in mice inoculated with a virulent parental strain (BH3479). Light microscopic examination revealed neuronal necrosis in the olfactory bulb and hippocampus of MVE-infected brains by 5 days post-infection (pi). Electron microscopy of these regions showed endoplasmic reticulum membrane proliferation, and tubular and spherical structures in the cisternae of the endoplasmic reticulum, Golgi complex and nuclear envelope. At seven to eight days pi, infected neurones exhibited chromatin condensation and extrusion, nuclear fragmentation, loss of segments of the nuclear envelope, reduced surface contact with adjacent cells and loss of cytoplasmic organelles. This cell injury was particularly noticeable in the proximal CA3 and distal CA1 regions of the hippocampus. The inflammatory cell profile consisted of macrophages, lymphocytes and especially neutrophils, and many of these inflammatory cells were apoptotic. High mortality rates in the BH3479-infected population of mice correlated with the intense polymorphonuclear and mononuclear leucocyte inflammatory infiltrate in the CNS.

In goldfish and frog, optic nerve regeneration is successful, with restoration of retinotopic projections in visual brain centres and the return of functional vision within 1–2 months. By contrast, at 1 year after unilateral optic nerve crush in the ornate dragon lizard (Ctenophorus ornatus), the regenerated retinotectal projections lack topographic order, presumably explaining why the lizards are blind via the experimental eye (Beazley et al. [1997] J. Comp. Neurol. 377:105–120). To determine whether other abnormalities are associated with the inability to restore topographic projections in the lizard, we charted anatomically the time course, accuracy, and stability of optic nerve regeneration by examining visual projections with the lipophillic dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) applied to the optic disk at intervals up to 1 year after optic nerve crush; in addition, DiI tracing of small groups of axons was used to examine the topicity of axons projecting to the tectum. Axons re-innervated visual centres from between 1 and 2 months, a time frame comparable with that in goldfish and frog. However, the projections in lizard were found to differ from those in goldfish and frog in three major ways. First, there was considerable variability within the projection patterns both between individual lizards at any one stage and with time. Second, the projections were inaccurate. As in normal lizards, the major projection was to the contralateral optic tectum, although it lacked detectable retinotopic axon order throughout. Furthermore, misrouting occurred such that regenerating axons formed a persistent projection to the ipsilateral side of the brain that was considerably stronger and more widespread than normal. Minor visual centres also became re-innervated but, in addition, regenerating axons formed persistent projections into the opposite optic nerve and to non-retino-recipient regions such as the nucleus rotundus, hypothalamus, and olfactory nerve, as well as the posterior and tectal commissures. Third, the projections appeared unstable. Projections to both tecta were strongest between 3 and 5 months, but they diminished thereafter. The results suggest that, compared with goldfish and frog, in lizards both pathway and target cues are degraded and/or cannot be read adequately; as a consequence, regenerating axons are unable to navigate exclusively to visual centres and cannot re-form stable connections. J. Comp. Neurol. 415:188–200, 2000. © 2000 Wiley-Liss, Inc.

The effect of aging on host resistance to systemic candidosis was assessed by monitoring the course of infection in 16-month-old CBA/CaH mice (aged non-immune) and in a comparable group that had been infected with a sublethal dose of Candida albicans at 6 weeks of age (aged immune). Aged non-immune mice showed rapid progression of the disease, with a marked increase in the number of mycelia in the brain and kidney, and early morbidity. Foci of myocardial necrosis were evident, but inflammatory cells were sparse. The histological picture in the aged immune mice was similar to that in the aged non-immune group, although fewer mycelial aggregates were seen. Both groups of aged mice showed a significantly lower fungal burden in the brain on day 1 of infection, but on day 4, colony counts increased significantly in the aged non-immune mice. Comparison of cytokine gene expression in the infected brains showed that the relative amount of interferon-gamma and tumour necrosis factor-alpha cDNA were similar in all three groups. Interleukin-6 was elevated in both infected non-immune and uninfected aged mice. Aged immune mice showed no morbidity after challenge, and both colonisation and tissue damage were reduced in comparison with the aged non-immune animals.

The conventional view of the effects of 1,25- (OH)2D3on osteoclastogenesis and bone resorption are thought to be mediated by stromal cells or osteoblastic cell. Giant cell tumor of bone (GCT) is characterized by multinuclear giant cells (osteoclast-like cells) distributed among a mass of mononuclear cells. Because it is very difficult to obtain normal human osteoclasts, many investigators, including ourselves, have used GCT as a source of human osteoclasts to enhance the understanding of normal skeletal remodeling and especially the hormone regulation of osteoclast functions. In this report, expression of human vitamin D receptor (VDR) mRNA was examined in the various components of GCT using the fluorescence in situ hybridization (FISH) technique. The results showed that VDR gene transcripts were expressed in both multinuclear cells (osteoclast-like cells) and mononuclear macrophage-like cells but not in stromal-like tumor cells in two cases of GCT examined. Measurement of VDR gene transcripts in situ by confocal microscopy showed that the level of VDR mRNA in osteoclast-like giant cells is greater than that in macrophage-like cells (p < 0.05). These findings suggest that 1,25- (OH)2D3mediated osteoclastic bone resorption or osteoclastogenesis could at least, in part, occur by direct action on osteoclasts/their precursor cells.

We have developed a model of focal Langerhans cell depletion by mild heat injury and used it to investigate the mechanisms of Langerhans cell repopulation in the injured epidermis. The possibility whether repopulation occurred by recruitment of precursor cells from the circulation or dermis or, alternatively, by migration from the surrounding normal epidermis into the injured area was considered. Repopulation was studied by evaluating the pattern of Langerhans cell reappearance and calculating the rate of change in the density. Heat injury followed by whole-body irradiation with shielding of the injured skin was used to assess repopulation in the absence of bone marrow precursors. Using tritiated thymidine autoradiography, we also investigated whether the newly arrived Langerhans cells (be they from circulating precursors or surrounding normal epidermis) actually divide. The results showed that heat injury completely eliminated the Langerhans cells within the area delineated by the injury. Two hours after injury, the Langerhans cells were fragmented and 2 days later, they could not be detected. Regeneration of the epidermis occurred 2 days after injury and Langerhans cells reappeared scattered somewhat sparsely in the centre of the lesion on day 3. These cells were small and slender, bearing one or two short dendrites. As the dendrites increased in number and in length, the cells became similar morphologically and phenotypically to normal Langerhans cells. The rate of repopulation increased dramatically between days 5 and 7 and reached normal density on day 11. The pattern of Langerhans cell repopulation in the injured area and the lack of repopulation in the irradiated animals indicated that repopulation occurs by immigration of precursors from the circulation or dermis. There was no indication of migration of Langerhans cells from surrounding normal epidermis. Lastly, the newly arrived Langerhans cells failed to divide at the site of injury.

The role of T lymphocytes in host responses to sublethal systemic infection with Candida albicans was evaluated by mAb depletion of CD4+ and CD8+ cells from BALB/c and CBA/CaH mice, which develop mild and severe tissue damage, respectively. Depletion of CD4+ lymphocytes from BALB/c mice markedly increased tissue damage, but did not alter the course of infection. In CBA/CaH mice, depletion of CD4+ cells abrogated tissue destruction in both brain and kidney at day 4 after infection, and significantly decreased fungal colonization in the brain. However, the severity of tissue lesions increased relative to controls from day 8 onwards. A small increase in tissue damage was evident in both mouse strains after depletion of CD8+ cells. There were no major differences between days 4 and 8 after infection in cDNA cytokine profiles of CD4+ lymphocytes from either BALB/c or CBA/CaH mice. After passive transfer into infected syngeneic recipients, spleen cells from infected CBA/CaH mice markedly increased tissue damage when compared to controls, and also caused a significant increase in fungal colonization in the brain. A similar transfer in BALB/c mice increased the number of inflammatory cells in and around the lesions, but had no effect on the fungal burden in brain and kidney. The data demonstrate that both CD4+ and CD8+ lymphocytes contribute to the reduction of tissue damage after systemic infection with C. albicans, and that the development and expression of CD4+ lymphocyte effector function is influenced by the genetic background of the mouse.

Although it is generally accepted that tumour growth is angiogenesis dependent, little is known about the role of angiogenesis in the metastatic process. Recent evidence suggests that the angiogenic tyrosine kinase receptor KDR is pivotal in new vessel formation. To investigate, therefore, the association between new vessel formation in primary breast carcinoma and axillary lymph node metastasis we have used computer assisted video analysis to assess the vascular distribution as well as the level of expression of KDR in individual vessels in sections of invasive breast carcinomas, some of which had metastasized to the axillary lymph nodes and some that had not. We specifically assessed the frequency distribution, perimeter, area, and density of KDR positive vessels in the same sections of tumours. Our results show that in invasive mammary carcinoma KDR is expressed exclusively on the surface and cytoplasm of endothelial cells of approximately 71% of vessels, but the level of expression in individual vessels does not correlate with the presence of axillary lymph node metastases (P > 0.10). However, we found that small vessels express higher levels of KDR (P < 0.02) than larger vessels and that there is a significantly higher frequency of relatively small (< 90 μm in perimeter) KDR positive vessels in breast tumours that had metastasized to the axillary lymph nodes than those that had not (P < 0.001). In conclusion, increased density and frequency of KDR positive small vessels in primary invasive breast carcinoma correlates with axillary lymph node metastases.

The severity of systemic infection with the yeast Candida albicans has been shown to be under complex genetic control. C57/L mice carry an allele that is associated with an increase in tissue destruction when compared with C57BI/6 mice; however, the gene affects only the severity of tissue lesions, and does not influence the magnitude of the fungal burden in either kidney or brain. Studies in [C57/L x C57BI/6]F1 hybrid mice, and [C57/L x C57BI/6]F1 x C57/L backcross mice, demonstrated that the gene behaves as a simple Mendelian co-dominant. (C) 1998 Academic Press.

BACKGROUND: As human cytomegalovirus (HCMV) infections are implicated in insulin-dependent diabetes mellitus (IDDM), the effects of murine (M)CMV infection of inbred mice on the pancreas are of interest. RESULTS: Inflammation and periacinar oedema peaked on day 3 and were replaced by a focal inflammation, but infected cells were rare. The islets were spared in C57BL mice. Insulitis normally seen in non-obese diabetic (NOD) mice was accelerated, but infected NOD mice did not become glycosuric. Isotypes of total and autoreactive antibodies suggested a shift to a Th 1 response (IgG2a) in all MCMV-infected mice. MCMV-induced pancreatitis was not affected by MHC genes but was similar or less severe in BALB/c mice. As these lack the Cmv1 gene, which provides a protective natural killer (NK) cell response in C57BL congenic mice, the C57BL background may carry a pancreatitis susceptibility gene able to counter NK-mediated restriction of viral replication. Consistently, congenic mice expressing Cmvl on a BALB/c background did not display pancreatitis, unless depleted of NK cells. In vivo treatment with soluble cytokine receptors suggested that interleukin 1 (IL-1) and/or tumour necrosis factor alpha contribute to acinar necrosis in C57BL mice.

Giant cell tumor of bone (GCT) is one of a few neoplasms in which the macrophage/osteoclast precursor cells and osteoclast-like giant cells infiltrate the tumor mass. Monocyte chemoattractant protein 1 (MCP-1) is a potent chemotactic factor specific for monocytes. In search of relevant cytokines that may enhance the recruitment of these reactive cells, we evaluated the localization and regulation of MCP-1 mRNA and protein in GCT by using Northern blot analysis, in situ hybridization and immunohistochemistry. We also determined whether conditioned medium obtained from GCT cultures can recruit human peripheral blood monocytes (CD68+) in an in vitro chemotactic assay. Using Northern blot analysis, we detected the specific gene transcript for MCP-1 in all GCT samples tested. In situ hybridization and immunohistochemistry revealed that both MCP-1 gene transcript and protein were consistently present in the cytoplasm of stromal-like tumor cells of GCT. Treatment of mononuclear cells from GCT at third passage with TGF-β1 for 24 h increased the level of MCP-1 mRNA in a dose-dependent manner, with the maximum effect at 1 ng/ml. Conditioned media from GCT cultures promoted the chemotactic migration of CD68+ peripheral monocytes, an activity which was abolished by the addition of MCP-1 antibody to the conditioned medium. Thus, the results of this study suggest that recruitment of CD68+ macrophage-like cells may be due to the production MCP-1 by stromal-like tumor cells. These CD68+ cells may originate from peripheral blood and could have the capability of further differentiating into osteoclasts in the tumor. J. Cell. Biochem. 70:121–129, 1998. © 1998 Wiley-Liss, Inc.

Pax7 is a paired-type homeobox gene which has previously been shown to play an important role in skeletal muscle formation. It is expressed in skeletal muscle of the limbs during embryogenesis and in adulthood. The aims of this study were firstly to determine the degree of polymorphism of Pax7 amongst inbred laboratory mice using Southern blotting and Pax7 regional specific sub-probes. Secondly, functional studies were performed on mice with each of the different structural forms of Pax7 to determine whether they were associated with differences in the ability to regenerate damaged skeletal muscle. Four different allelic forms of Pax7 have now been identified in laboratory mice indicating that the previously reported DNA sequence of Pax7 is not applicable to all laboratory mice. Hybridisation patterns of TaqI digested DNA representing each of the different Pax7 alleles with the Pax7 specific sub-probes suggested that in contrast to previous findings, Pax7 is associated with two highly polymorphic homeoboxes. The presence of two homeoboxes in BALB/c mice has been confirmed by DNA sequencing. Results of functional studies have also shown that the ability to regenerate damaged skeletal muscle in adult mice is strongly associated with the presence of a 0.15-kb TaqI fragment derived from one of the homeoboxes.

The peritoneum is more than a mechanical covering that allows for the easy gliding of opposed peritoneal surfaces. The peritoneal mesothelial cells facilitate the action of powerful innate immune mechanisms. In addition, the peritoneal-associated lymphoid tissues contain unique cells that may play a crucial role in the localization of intraperitoneal infection. A clearer understanding of the molecular and cellular events underlying peritoneal functions in both the unstimulated and stimulated state will aid future treatment of peritonitis.

Two alternate allelic forms of human cytosine 5-methyltransferase, 5-MT I and 5-MT II, which differ by the absence or presence of an intronic MspI recognition sequence, have been recognised. The polymorphic region was localised using a series of subprobes prepared upon MspI digestion of the 2.5-kb cDNA probe (hmt-2.5). A PCR-based method was then developed for rapid 5-MT genotyping. The gene and phenotype frequencies of 5-MT I and 5-MT II were not significantly different in genomic DNA samples from a series of non-Hodgkin’s lymphomas and breast cancer cases compared with DNA from normal subjects. Allelism of 5-MT allows new approaches to the assessment of variation in gene copy number of 5-MT in different types of neoplasia.

Osteoclasts resorb bone by a complex dynamic process that initially involves attachment, polarization and enzyme secretion, followed by their detachment and migration to new sites. In this study, we postulated that mineralized and osteoid bone matrix signal osteoclasts differently, resulting in the resorption of mineralized bone matrix only. We, therefore, compared the cytoplasmic distribution of cytoskeletal proteins F-actin and vinculin using confocal laser-scanning microscopy in osteoclasts cultured on mineralized and demineralized bone slices and correlated the observations with their functional activity. Our results have demonstrated significant differences in F-actin and vinculin staining patterns between osteoclasts cultured on mineralized bone matrix and those on demineralized bone matrix. In addition, the structural variations were accompanied by significant differences in bone resorbing activity between osteoclasts grown on mineralized bone matrix and those on demineralized bone matrix after 24 h of culture --resorption only occurring in mineralized bone but not in demineralized bone. These results indicated that failure of osteoid bone resorption is caused by perturbation of osteoclast polarization.

Case-control methodology was used to evaluate the significance of vascularity in small breast carcinomas with regard to the presence or absence of axillary lymph node metastases. Vascularity was assessed in 32 axillary node positive primary breast tumours (LN+ve) less than 2 cm in size and compared with 56 control axillary node negative primary tumours (LN-ve), which were matched for histological type and grade and tumour size. This study design employed computer-assisted video analysis (CAVA) to assess the total blood vessel perimeter (BVP), total blood vessel area (BVA), and total blood vessel density (BVD) throughout a tissue section that encompassed an entire cross section of the tumour and its immediate periphery. The BVA and BVD in these tumours were not significantly different between LN+ve and LN-ve groups. The LN-ve carcinomas had, on average, a significantly (P0.05) higher total BVP (3355 microm/mm2) than LN+ve tumours (2771 microm/mm2). 'Hot spot' areas were also independently assessed by two pathologists and the same areas measured by CAVA. A strong correlation (P0.001) between the two methods of assessment of BVD of the neovascular 'hot spots' was found; however, no association with axillary lymph node metastasis was found using either method of assessment. In conclusion, vascularity assessed by either blood vessel density or blood vessel size in primary invasive breast cancers less than 2 cm in diameter showed no association with axillary lymph node metastasis; in fact a negative association was found with total BVP of whole tumour sections and BVD in 'hot spots' using CAVA. Further, this study has established a computer-assisted method of quantifying vascularity in solid neoplasms and is a positive step towards a standardised approach to this diverse and methodologically variable area.

Ribotyping and field alternation gel electrophoresis (FAGE) were used to examine 19 Pasteurella multocida isolates, and to assess the ability of these techniques to differentiate P. multocida strains that cause haemorrhagic septicaemia (HS). Reproducible patterns were obtained from both methods, with FAGE demonstrating greater discriminatory power than ribotyping. FAGE analysis was particularly useful in distinguishing North American cultures originating from the 1922 Yellowstone National Park Buffalo 'B' strain, demonstrating the ability to detect genetic alterations induced by repeated subculture. A remarkable homogeneity was observed among Asian HS strains following ribotyping and FAGE analysis, with a clear distinction observed between virulent and avirulent HS isolates. This study has illustrated the value of genomic fingerprinting methods in distinguishing strains of similar serotype, and the capability of these methods to produce detailed characterisation of P. multocida isolates.

Amplification of multiple P multocida genomic DNA fragments by outwardly-directed primers based on the repetitive extragenic palindromic (REP) consensus sequence, generated complex profiles in a PCR-based fingerprinting method known as REP-PCR. Polymorphisms within REP-PCR profiles were used to characterise 38 isolates of P multocida. The high degree of homogeneity observed among haemorrhagic septicaemia (HS) strains of serotype B and E provided evidence of a disease-associated REP profile that may serve as a novel method for the identification of HS strains regardless of serotype. REP-PCR profiles of other P multocida serotypes were highly variable, illustrating the potential of this technique for the molecular fingerprinting of fowl cholera or atrophic rhinitis isolates. A specific amplified REP fragment was isolated and used to probe membrane-bound digested P multocida genomic DNA. Hybridisation patterns not only distinguished HS-causing isolates from non-HS P multocida, but also demonstrated a degree of relatedness between HS and HS-like strains.

The hippocampal region of apolipoprotein E-deficient mice of varying ages was examined for any morphological changes by light and electron microscopy. Unusual periodic acid-Schiff-positive granules were seen in the hippocampal area of these animals as early as the fourth week of life and their numbers increased gradually with age. These granules were never found in control C57BL/6J (B6) mice before six months-of-age and their numbers were invariable low. They were strongly congophilic when stained with a modified Congo Red technique and reacted with a monoclonal antibody specific to amino acids 17-24 and 35-43 of the beta-amyloid peptide. The immunostaining of these granules with the beta-amyloid peptide was lost after specific adsorption with the appropriate synthetic peptide. These granules were identified ultrastructurally as non-membrane-bound fibrillogranular material in the cytoplasm of protoplasmic astrocytes. The data indicate that an amyloid-like protein accumulates in the protoplasmic astrocytes of the hippocampus of apolipoprotein E-deficient mice, especially in the brains of old animals.

Tissue damage in the kidney and brain after systemic infection with Candida albicans was examined in recombinant inbred strains (AKXL) derived from AKR and C57/L progenitors. Nine of the 15 strains showed mild (C57/L-like) tissue damage. Of the remainder, two strains developed lesions comparable to the AKR parental strain, whereas four exhibited a much move severe pattern of tissue damage. This was characterized by pronounced mycelial growth in the brain, and gross oedema of the kidney, with extensive fungal colonization and marked tissue destruction. The presence of the null allele of the haemolytic complement gene (Hc) may be necessary but not sufficient, for the expression of the very severe lesions. The results were interpreted as reflecting the actions of two independent genes, which have been designated Carg1 and Carg2 (Candida albicans resistance genes 1 and 2). (C) 1997 Academic Press Limited.

Background At the advent of laparoscopic adrenalectomy it seemed timely to us to assess the advantages and the overall results of the different techniques that are currently used in an approach to adrenalectomy. Patients and methods Between 1984 and 1995, 165 patients underwent adrenalectomy. Eightysix patients (37 men and 49 women with a mean age of 46.4 years) underwent adrenalectomy via the anterior approach, 61 patients (18 men and 43 women with a mean age of 43.8 years) underwent posterior extraperitoneal adrenalectomy, and 18 patients (8 men and 10 women with a mean age of 48.7 years) underwent anterior laparoscopic adrenalectomy. For statistical analysis of the different comparisons between the groups we used the t test for independent samples, the Wilcoxon test, chi-square, and one way analysis of variance. Results There was no operative mortality. The morbidity was 13.9% in the anterior approach, 9.8% in the posterior approach, and 0% in the laparoscopic approach. The mean operating time for unilateral adrenalectomy was 155.3 min (range 75 to 315) for the anterior approach, 108.6 min (range 60 to 195) for the posterior approach and 116.1 min (range 75 to 180) for the laparoscopic approach. For bilateral adrenalectomy the mean operating time was 165 min for the anterior and 178 min for the posterior approach. The average diameter of tumors resected anteriorly was 8.07 cm (range 2.5 to 20), posteriorly was 5.25 cm (range 0.5 to 14), and laparoscopically was 4.03 cm (range 2 to 6.5). The mean length of postoperative hospitalization for patients undergoing unilateral adrenalectomy was 8 days (range 2 to 25) for the anterior approach, 4.5 days (range 1 to 11) for the posterior approach, and 2.2 days (range 1 to 5) for the laparoscopic approach. Patient controlled analgesia lasted 3.4 days for those operated anteriorly, 2.3 days for those operated posteriorly, and 1.08 days for those that underwent laparoscopic adrenalectomy. Conclusions The laparoscopic approach to the adrenal promises the safest and least painful operation with shorter in-hospital stay and the best cosmetic and long-term results. The posterior approach is the fastest of all and a better overall operation than the anterior approach that should only be reserved for removing very large adrenal tumors and when concomitant intra-abdominal procedures, that can't be handled laparoscopically, are anticipated.

Microsatellite instability is a form of genetic damage that may be due to defective mismatch repair genes and may be a marker of processes leading to malignancy. We have analysed a series of epithelial hyperplasia of usual type, carcinomas in situ and invasive and metastatic carcinomas from the mammary gland on the assumption that they represent stages in the evolution of mammary carcinoma. Eight markers on chromosomes 3p, 4q, 9p, 11p, 14q, 17p, 17q and Xq were examined for microsatellite instability and loss of heterozygosity. High rates of loss on chromosomes 17p, 17q and Xq indicate that these chromosomal arms contain genes important in mammary carcinogenesis. The rate of microsatellite instability observed in this study was uniformly low, irrespective of the lesion. This implies that microsatellite instability is not a marker of malignancy in most instances of mammary neoplasia. Images Figure 1 Figure 2 Figure 3

Objective: To describe the practice of electroconvulsive therapy (ECT) in Greece. Methods: A survey was conducted during the academic year 2008-2009. Electroconvulsive therapy use was investigated for 2007. All civilian institutions providing inpatient care were included. Centers that provided ECT completed a 57-item questionnaire. Centers that did not offer ECT completed a 13-item questionnaire. Results: Fifty-five (82.1%) of 67 institutions responded. Electroconvulsive therapy was offered in 18 hospitals. Only 2 of 10 university hospitals offered ECT. Overall, 137 patients were treated with 1271 sessions in 2007. Only 1.47% discontinued treatment owing to adverse events. There were no deaths. Schizophrenia was the most common diagnosis (41.3%) among those receiving ECT, followed by major depression (28.9%), bipolar depression (9.1%), catatonia (4.1%), suicidal ideation (3.3%), and schizoaffective disorder (2.5%). Physicians considered major depression (93.8%), catatonia (86.5%), schizophrenia (56.3%), and mania (50%) the most appropriate indications. Written informed consent was required in 77.8% of the institutions, whereas the rest required verbal consent. Bilateral ECT was the preferred electrode placement (88.9%). Modified ECT was used exclusively. Propofol was the preferred anesthetic (44.4%), followed by thiopental (38.9%). Seven (38.9%) of 18 hospitals used a fixed stimulus dose at first treatment. Five (27.8%) of 18 hospitals used the half-age method. Continuation/maintenance ECT was used in 33.3% of the hospitals. Outpatient ECT was seldom used. Lack of training, difficult access to anesthesiology, billing issues, and stigma were cited as the main impediments to the practice of ECT. Conclusions: Electroconvulsive therapy is practiced in moderate numbers in Greece and almost exclusively on an inpatient basis. Lack of training and lack of availability of anesthesiologists were cited as the most common obstacles to providing ECT.

The fate of dividing mouse mammary epithelial cells was followed by use of tritiated thymidine (3H-Tdr) autoradiography. Loss of label consistent with halving kinetics was observed at various times after injection; however, heavily labelled cells were frequently observed at two weeks and later, when none was expected. The grain count over these heavily labelled cells was often comparable with that 1 h after 3H-Tdr injection. Extensive serial sectioning revealed that the heavily labelled cells were often single cells surrounded by many unlabelled cells or that their label was in stark contrast (in excess of 20 reduced silver grains) to the surrounding group of cells whose label was just above background (a maximum of 3 grains). In addition, by injecting mice at different stages of oestrus, we demonstrated that these long-lived cells, although influenced by oestrus, replicated independently of the oestrogen peak. Our data support a model for mouse mammary epithelium that has a single 'stem' cell positioned within a group of its progeny to form a discrete proliferative unit. This model requires many such stem cells within the mammary epithelium and is consistent with similar models proposed for other tissues.

The luminal plasmalemma of regenerating mesothelial cells was examined by transmission electron microscopy and the concentration of microvilli at various stages of healing was quantified. Charged tracer and lectin binding techniques were also employed to investigate electrostatic and chemical changes in mesothelial glycocalyx. In uninjured mesothelium and at all stages of healing, the concentration of microvilli at the cellular periphery was greater than over the main cell mass (P < 0.05). Furthermore, there was an increase in the concentration of microvilli in all regions by day 4, which reached a maximum at day 6, then at days 10-15 returned to values closer to uninjured mesothelium (P < 0.01). These changes were associated with an alteration in surface charge. In all lesions, the surface charge on microvillar membranes was greater than for flat membranes, but the difference was only significant at days 4, 6, and 15 (P < 0.001). The changes in surface charge may reflect a differential expression of mucopolysaccharides on the surface membrane. In addition, concanavalin A bound avidly to mesothelial surface membranes, suggesting the presence of alpha-methyl-D-mannoside residues. These findings suggest an association between microvillar formation and surface charge, the former protecting the healing mesothelium by enhancing entrapment of serosal fluid and its contents.

This paper describes the first successful in vivo application of antisense DNA technology to induce the accumulation of photoreceptor outer segment derived debris in the retina. An antisense oligonucleotide (CatSC), which was previously demonstrated to be an effective tool to induce debris accumulation in vitro, was injected into the vitreous of pigmented and non-pigmented rats. The animals were euthanased 7 days after the injections. The number of inclusions significantly increased in the RPE layer of Long Evans and RCS-rdy + rats injected with 66 ug of CatSC to 96.13 ↣ 2.6 (SD) (p < 0.0003) and 204.2 ↣ 39.3 (SD) (p < 0.0001), respectively. The difference between the number of phagosome-like inclusions present in control saline, 6.6 ug of CatSC or 66 ug of sense oligonucleotide (S1) injected animals was not statistically significant. There were no abnormalities observed in the inner layers of the retina but the accumulation of phagosome-like inclusions was accompanied by disorganisation in the apices of ...

Summary Previous studies have demonstrated upregulation of intestinal mucosal macrophage CD16 (an Fc receptor for IgG) in bone marrow transplant (BMT) recipients with graft-versus-host disease (GVHD). We sought to determine whether there was ultrastructural evidence of mucosat macrophage activation in allogeneic BMT recipients and relate appearances to those seen in autologous BMT patients and to immunohistological findings. Sigmoid colonic mucosal biopsies from five allogeneic and three autologous BMT patients were taken prior to, 30 days after transplant and, in three of the allogeneic patients, 120 days after transplant. These were examined by immunohisto-chemistry and electron microscopy. Immunohistological analysis revealed upregulation of lamina propria macrophage CD16 after transplant in all patients except one autologous BMT recipient; there were no such changes in total macrophage numbers. Ultrastructural evidence of lamina propria macrophage activation was prominent after both allogeneic and autologous BMT. There was an increase in nuclear size accompanied by increased euchromatin and larger nucleoli. In the cytoplasm there were increased numbers of lysosomes, many of which were small and cylindrical, and cytoplasmic flaps were prominent. Phagosomes were less numerous after transplant. These data confirm that after BMT intestinal mucosal macrophages become activated. However changes in macrophage uttrastructure specific to patients at risk of developing clinical GVHD are lacking.

The yeastCandida albicansis an important opportunistic pathogen that has been associated with disease of the inner ear. This study describes the histopathology of acute labyrinthitis caused by systemic infection with C.albicansin ageing inbred mice. Within four days after infection, yeast and hyphal forms of C.albicanswere found in the membranous labyrinth. The utricle and the adjacent parts of the ampullary regions of the semicircular canals were most severely affected, but damage was also seen in the scala media, the Scala tympani, the saccule, and the scala vestibuli. In the utricle, the lining epithelium of the membranous labyrinth was disrupted, and the lining cells of the vestibular membrane showed foci in which the membrane was disrupted. The data suggest that age may represent a risk factor for fungal labyrinthitis.

This study determined whether the genotype of bone marrow-derived inflammatory cells contributes to the more pronounced leukocytic exudation and extensive new muscle formation seen in SJL/J compared with BALB/c mice after a crush-injury (Mitchell et al. 1992). Female SJL/J mice were whole-body irradiated and reconstituted with male bone marrow from the BALB/c strain, and irradiated BALB/c females reconstituted with male SJL/J bone marrow. The mice were allowed to recover for 3 weeks and the tibialis anterior muscle (in a leg which had been protected from irradiation) was injured by crushing. At 3 and 10 days after injury the extent of necrotic debris, mononuclear leukocytic infiltration and new muscle formation was assessed in the muscles. The SJL/J mice reconstituted with BALB/c bone marrow showed extensive mononuclear leukocytic infiltration and clearance of necrotic debris when compared with BALB/c mice reconstituted with SJL/J bone marrow, and these strain-specific differences mirrored those seen with control bone marrow reconstituted hosts and non-irradiated hosts. The results show that the genotype of the bone marrow-derived macrophages is not responsible for the superior regeneration of crush-injured skeletal muscle in SJL/J mice, and it appears that factors intrinsic to the muscle tissue may be of central importance.