Introduction
============

DNA methylation is involved in cellular development, differentiation and transformation \[[@B1]\]. In different types of tumours, aberrant methylation of CpG islands in the promoter region has been observed for many differentiation- and cancer-related genes resulting in the silencing of their expression \[[@B2]\]. Therefore, over the past decade, there has been increasing interest in the use of demethylating agents to induce the differentiation or the apoptosis of cancer cells \[[@B3],[@B4]\]. Treatment of the cells with the pyrimidine analogue 5-AzaCytidine (AzaC), which inhibits methylation of cytosine residues during replication in the newly synthesised DNA, has been demonstrated to reactivate the expression of many silenced genes, as well as the expression of the silenced retro viral genomes \[[@B5]\]. Silencing of one of the most important cell cycle regulatory proteins p16^INK4a^ by methylation of the CpG islands in the promoter region has been found to be a common event in tumours \[[@B6],[@B7]\]. Protein p16 suppresses S-phase entry by antagonising the cyclin-dependent kinases CDK4 and CDK6 \[[@B8]\].

Deciphering the molecular mechanisms underlying the phenotypic effects of the treatment with demethylating drugs is a crucial step in understanding what genes may be interesting targets for chemotherapy. The available data on the mechanism of action of these drugs strengthen the idea that it is different from that of agents that act primarily via their cytotoxic effects, such as Arc-C \[[@B9]\]. Several lines of evidence suggest that galectin-1 (Gal-1), a 14 kDa galactoside-binding protein distributed widely in immune cells, could be involved in these mechanisms. Several members of the galectin family have been found to modulate cell differentiation and cell survival \[[@B10]-[@B15]\]. Early studies demonstrated that the expression of Gal1 could be induced in cultured hepatoma-derived cells by treatment with AzaC \[[@B16]\]. Chiariotti and co-workers showed that reactivation of the silent Gal1 alleles is accompanied by a transition from a fully methylated to a fully unmethylated state of several CpG dinucleotides in the promoter region \[[@B17]\]. In addition, nonexpressing tissues exhibited highly heterogeneous methylation profiles \[[@B18]\]. Gal is considered to be a typical cytosolic protein, lacking a signal peptide for membrane translocation \[[@B19]\]. However, most of the functions assigned to galectins are confined to the cell surface or extracellular milieu \[[@B10],[@B20],[@B21]\], consistent with evidences for extracellular roles of Gal1 in regulation of cellular differentiation and proliferation. It is clear that Gal1 can be specifically secreted and targeted by an infrequent mechanism \[[@B22]-[@B24]\]. The constitutive expression as well as the secretion of Gal1 dramatically depend on cell types \[[@B25]\] and are responsive to developmental events \[[@B20],[@B22],[@B23]\].

An example is found during erythroid differentiation of the K562 human leukaemia cell line. During differentiation induced by erythropoietin and deprivation of granulocyte-macrophage colony-stimulating factor, the cells empty their cytoplasmic content of endogenous Gal1 into the external medium where it is bind to cell surface receptors \[[@B24]\]. The synthesis and secretion of Gal1 by leukocytes are of interest because lactosaminoglycans present at the leukocyte cell surface may be physiologically significant galectin receptors that could mediate autocrine or paracrine functions. Several lines of evidence indicate that Gal1 may function as an autocrine negative growth regulator or as a pro-apoptotic factor \[[@B26]-[@B28]\]. We have recently demonstrated that Gal1 binding to Burkitt lymphoma cells results in an intracellular signal, with inhibition of the tyrosine phosphatase activity of CD45 and therefore phosphorylation of Lyn kinase \[[@B29],[@B30]\].

In this work, we study the effect of AzaC treatment on the lymphoma cell line BL36. As p16^INK4a^ gene has been found to be downregulated by hypermethylation at high frequency in different types of tumours \[[@B6],[@B7]\], it is used as a control of AzaC effect. Then, the phenotypic effects of AzaC are compared to those obtained by the addition of exogenous Gal1. The findings that we report here lend further support to a potential role for Gal1 in the AzaC-induced pathway of differentiation in hematopoietic cells.

Materials and Methods
=====================

The BL36 B lymphoma cell line \[[@B31]\], a gift from Pr. Lenoir (CIRC, Lyon, France) to Pr. M. Raphael, was maintained in a complete medium of RPMI 1640, containing 10% heat-inactivated foetal calf serum, 2 mM L-Glutamine, 1 mM sodium pyruvate (complete medium). Treatment with AzaC was conducted as follows: 24 hours after seeding 5, 10 or 50 μM of AzaC were added to 5 ml of complete medium \[[@B16],[@B32]\]. The cells were feeded with the appropriate medium during the time of the experiments.

Several independent methods were used to assess the proliferative versus death states of the cells. For direct determination of cell number, at the indicated time point the cells were harvested and counted using a Coulter Counter (Beckman Coulter France, Villepinte, France). Determination of the viable cell numbers used propidium iodide (PI) (Sigma, Saint-Quentin Fallavier, France). To determine the percent cell death including both apoptotic and necrotic cells, AzaC-treated cells were analyzed for AnnexinV binding and PI uptake as described \[[@B33]\] using the ApoDetect AnnexinV-FITC kit (Zymed). Briefly, after washing of cultured cells (3.10^5^) with PBS and resuspension in binding buffer, the cells were stained with 10 μl FITC-labelled AnnexinV and 10 μl PI (20 μg/ml). After 5 min of incubation at room temperature in the dark, again 200 μl of binding buffer was added and cells were analyzed by flow cytometry. The percent cell death was calculated by determining the percent of viable cells:

The MTS assay (CellTiter 96™, Promega, Charbonnières, France) \[[@B34]\] was used to compare the percentage of metabolically active cells in treated cells vs. untreated cells, as previously described \[[@B14]\]. To study the cell cycle, 2.10^6^ BL36 cells were pelleted at 1000 g for 5 minutes and the pellet was incubated in a mixture of ethanol-PBS (70/30 v/v). The cells were pelleted another time and suspended in 800 μl PBS. One hundred microliters of 1 mg/ml RNase was added to the cell suspension. One hundred microliters of 400 μg/ml PI was added to the solution to stain the nuclear DNA. The DNA content of the cells was determined by a flow cytometer, FACScan (Becton Dickinson, Mountain View, CA), and the percentages of cells in G0+G1, S, and G2+M phases of the cell cycle were analyzed by a polynomial model (SFIT, Becton Dickinson).

To determine the cell phenotypes, cells were suspended in PBS supplemented with 2% BSA, and incubated in suspension for 30 min with the fluorescently tagged primary antibody or negative control, or incubated with untagged antibody, washed, and incubated again for 30 min with FITC-conjugated second antibody. All incubations were performed at 4°C. Flow cytometry was performed using a Coulter Epics Elite ESP instrument.

Human recombinant Gal1 (rGal1) was obtained as described elsewhere \[[@B14]\]. The protein was purified by affinity chromatography on a lactosyl-divinylsulfone-agarose column. Antibodies against human Gal1 were generated as described in \[[@B35]\] and \[[@B36]\]. The immunolocalization of Gal1 was carried out as described for K562 cells \[[@B24]\].

For the preparation of cell extracts, a total of 17.10^6^ cells were solubilized in 1 ml of extraction buffer: 50 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% SDS (w/v), 0.5% sodium deoxycholate (w/v), 0.5% Nonidet NP40 (v/v). Finally, 1 tablet of antiprotease cocktail (Roche, Meylan, France) was added to 10 ml of buffer. The cells were sonicated in ice three times. The lysat was centrifuged at 100, 000 g for 15 min at 4°C. The supernatant was collected and stored at -20°C until used. Proteins were resolved by discontinuous SDS-PAGE on 1.5 mm gel (T: 6--18%) according to the method of Laemmli. \[[@B37]\]. For Western blotting, proteins separated by SDS-PAGE were electrotransferred onto Immobilon-P membrane (Millipore). Blots were incubated with 1:50 anti-Gal1 mAb, or 1:500 anti-pl6 mAb (Pharmingen) for 2 h at room temperature. The blots were developed with anti-mouse Ig antibody-HRP diluted 1/10,000, followed by incubation in the Amplified Opti-4CN kit substrate (Bio-Rad). The image of the membranes was acquired from GS-700 Densitometer 4, and analyzed with Molecular Analyst Software (Bio-Rad).

Results
=======

During the four days of treatment with AzaC, cell proliferation appeared to slow (Fig. [1A](#F1){ref-type="fig"}). When BL36 cells were cultured with AzaC at the high concentration of 50 μM, cell proliferation was strongly inhibited. BL36 cells exposed to 5 μM AzaC also exhibited a significant reduced growth rate but these cells were \>95% viable by propidium iodide test even at day 4 (data not shown), and were found metabolically active by MTS assay as described below. According to these results, the following experiments addressing metabolic activity, death, and cell differentiation were performed.

![Effect of AzaC and Gal1 on growth and viability of BL36 cells in culture. A. The cells were seeded at 4 × 10^5^/ml and exposed to AzaC at various concentrations for zero to 4 days. Cells were counted every day on a Coulter Counter. B. Cells were cultured in triplicate in the presence or absence of AzaC. Metabolic activity was measured at the indicated times by the MTS assay. Results are given as mean percentage of metabolically active cells (%) in cells grown in the presence of AzaC compared to cells grown in the absence of AzaC. C. Cells grown in the presence of various concentrations of Gal1 were compared to control cells. The data represent the mean of 3 experiments.](1475-2867-1-2-1){#F1}

The proportion of metabolically active cells was determined using the MTS test in cultures treated with AzaC in comparison with untreated cells (Fig. [1B](#F1){ref-type="fig"}). The cellular conversion of MTS to the ultraviolet-absorbing formazan product has been demonstrated to be directly proportional to cellular metabolism resulting in the formation of reducing equivalents such as NADH or NADPH \[[@B38]\]. A drop in the % of metabolically active cells of about 36% was observed over the 24 h period following the addition of 10 μM AzaC, and went up to 42% in cells treated with 50 μM AzaC. Cell death was confirmed by an increase in annexinV/PI staining (Fig. [2](#F2){ref-type="fig"}). Within 48 h of incubation with AzaC at 10 μM, the number of AnnexinV^+^ PI^-^ apoptotic cells and AnnexinV^+^ PI^+^ necrotic cells increased. For longer incubation times, induced cell death increased dramatically.

![Induction of cell death in cells treated by either AzaC or rGal1. The BL36 cell line was treated with 10 μM AzaC, 700 nM rGal1, or buffer control and the percent cell death was evaluated by annexinV/propidium iodide staining, as described in \"Materials and Methods\". There is a dramatic increase in annexinV^+^/propidium iodide^+^ cells in the BL36 samples treated with AzaC, not seen with the cells treated with Gal1.](1475-2867-1-2-2){#F2}

Studies investigating the mechanisms whereby B lymphoma cells are induced to undergo apoptosis demonstrated that an arrest in the cell cycle preceded apoptosis \[[@B39]\]. To determine whether AzaC modified the cell cycle distribution of BL36 cells, the DNA content of AzaC-treated cells was analyzed by PI staining. Cells were exposed to 10 μM AzaC for 2, 4 and 7 days. After 7 days, AzaC-treated cells exhibited a relative increase of cells in G0/G1 (77 %) in comparison with controls (56%). In the same time, the percentage of cells in the S phase compartment drops from 38 to 18%, suggesting an accumulation of cells into the G1 phase of the cell cycle (Fig. [3](#F3){ref-type="fig"}). However, there was no change in the relative cell cycle distribution at 2 or 4 days after AzaC treatment. By this time, a significant population of cells has undergone apoptosis. This suggests that, at least during the first days of treatment, inhibition of growth is uncoupled with an arrest in the cell cycle.

![Cell cycle analysis of AzaC-treated and control cells. The BL36 cells were incubated for 7 days in the presence of 10 μM AzaC and the DNA contents were measured.](1475-2867-1-2-3){#F3}

The effects of different concentrations of AzaC (5, 10, and 50 μM) on the expression of various surface antigen of BL36 cells were investigated. AzaC increased the cell population that expressed the cell-surface maturation marker CD23, CD30 present on activated B-lymphocytes \[[@B40]\] and CD138 (Syndecan-1). After the treatment with 50 μM AzaC some changes were observed that were not detected at lower concentrations: an increase of the cell population that expressed CD21, and a decrease of the cell population that expressed CD19 that is lost on maturation to plasma cells \[[@B41]\], and CD71 that is expressed on proliferating cells \[[@B42]\] (Table [1](#T1){ref-type="table"}).

::: {#T1 .table-wrap}
::: {.caption}
######

Modulation of urface Markers on BL36 Cells by AzaC
:::

  Surface marker   Control   Treatment AzaC = 5 μM   AzaC- 10 μM   AzaC = 50 μM
  ---------------- --------- ----------------------- ------------- --------------
  CD 19            94.9      88.5                    86.1          58.1
  CD 21            \<20      \<20                    \<20          20.3
  CD 23            31.5      47.6                    45.2          63.8
  CD 30            \<20      \<20                    48.4          21.5
  CD 45 RA         76.7      74.4                    71.2          64.6
  CD 45 RO         21.4      35.1                    33.3          38.5
  CD 71            97.3      94.9                    93.8          83.8
  CD 138           20        23.9                    23.8          24.8

BL36 cells were exposed with 5 μM, 10 μM and 50 μM AzaC for 4 days. Then cell surface antigens were analyzed by flow cytometry. Values below 20% were considered as negative. In the same conditions, CDs 3 and 10 remain negative, and no variability was observed for CDs 11 a, 18, 25, 38, 44, 48, 54, 58, 77, 80, and 95.
:::

P16 is a biochemical marker of cell cycle progression \[[@B43]\] and is also commonly utilized as a marker of demethylation reaction \[[@B44]\]. Consequently, we examined the effects of AzaC treatment on expression of p16 in BL36 cells. As expected, when these cells were treated with AzaC for 48 h, we detected the presence of p16 by Western Blotting (Fig. [4A](#F4){ref-type="fig"}). Thus, taken together the data showed that AzaC treatment induced an inhibition of cell growth related to an arrest at G0/G1 phase of the cell cycle, and confirmed the efficiency of AzaC treatment for expressing the tumour suppressor gene p16.

![Immunoblotting of p16 and Gal1 from extracts of BL36 treated by AzaC. After different periods of treatment by AzaC, cell extracts were separated by SDS-PAGE, followed by electrophoretic transfer onto Immobilon-P membranes and immunostaining with (A) anti-pl6, or (B) anti-Gal1 monoclonal antibodies. Hela cells extract and rGal1 were used as positive controls for p16 and Gal1, respectively. (C) Representation of the variation of Gal1 expression in cells treated different times with 5 or 10 mM AzaC, using the analysis of the Western blots in Molecular Analyst software.](1475-2867-1-2-4){#F4}

To determine whether AzaC is able to induce the expression of Gal1 in B lymphoma cells, protein extracts prepared form cells treated with 5--10 μM AzaC and from untreated cells were separated by SDS-PAGE and analyzed by immunoblotting with anti-Gal1 antibody. Fig. [4B,C](#F4){ref-type="fig"} shows the rate of variation in Gal1 expression induced by the differentiating agent. The galectin was not detected either in untreated BL36 cells or BL36 cells treated with AzaC for 24 or 48 h. The galectin became detectable in AzaC-treated cells by 96 h. Then, to determine whether the newly synthesised galectin molecules in AzaC-treated BL36 cells are expressed on the cell surface, the binding of anti-Gal1 Ab to untreated and treated BL36 cells was studied by immunocytochemistry. To visualize only extracellular Gal1, cultures of BL36 cells were incubated with anti-Gal1 antibody without permeabilization of their membranes. Over the 4-day time-course, AzaC-induced Gal1 did progressively localise to the cell surface. However, Gal1 did not exclusively localise to the cell surface but was also distributed throughout the cytoplasm (Fig. [5](#F5){ref-type="fig"}).

![Immunoreactive localization of Gal1 in AzaC-treated BL36 cells. Cytospins were incubated with anti-Gal1 serum and peroxidase-stained (original magnification 1250×). First line: negative controls performed on untreated BL36 cells. No Gal1 was detectable with anti-Gal1 antibody. Second line: permeabilized cells treated cells stained with monospecific anti-Gal1 antibody; arrows indicate the localization of Gal1. Third line: after 144 h, Gal1 was detectable on the cell surface of nonpermeabilized cells. No immunostaining was observed with controls performed with preimmune serum (not shown).](1475-2867-1-2-5){#F5}

Several of the changes in AzaC-treated BL cells might be attributed directly to Gal1 induction, as Gal1 has been implicated in differentiation and growth inhibition \[[@B11]-[@B13],[@B29],[@B45],[@B46]\]. A series of experiments were thus performed to address whether changes observed in BL36 were direct AzaC effects and/or secondary effects related to Gal1 expression induced by AzaC treatment. To this end, BL36 was treated with recombinant Gal1 (rGal1). Slower growth rates were confirmed for the rGal1-treated cultures. A drop in the % of metabolically active cells of about 28% was observed over the 24 h period following the addition of 700 nM rGal1. However the proportion of metabolically active cells in cultures treated with rGal1 vs. the proportion in untreated cultures was not modified after 4 days of treatment (Fig. [1C](#F1){ref-type="fig"}). Moreover the variation of cell death detected in treated-cells relative to the control cultures was discrete (\<10%) and limited to the first day after the treatment (Fig. [2](#F2){ref-type="fig"}). To determine whether the inhibition of proliferation could be correlated to a modified phenotype, the effects of 700 nM rGal1 on the expression of various surface antigens shown to be modified or not by AzaC were investigated. The populations of cells that expressed CD19, CD23, CD45RO and CD45RA were unchanged. On the other hand, 24 h rGal1 treatment increased the cell population expressing CD138 (Fig. [6](#F6){ref-type="fig"}), a marker for plasma cells, while it decreased the expression of CD71. These modifications were reinforced after 48 h rGal1 treatment (Table [2](#T2){ref-type="table"}).

![Modification of the expression of CD138 in rGAL1-treated cells. Cells incubated for 24 or 48 h with rGal1 (700 nM) were stained with anti-CD138 antibody and analyzed by flow cytometry. Controls were performed in the absence of rGAL1. Representative-histograms: x-axis, log of the fluorescence intensity; y-axis, number of events.](1475-2867-1-2-6){#F6}

::: {#T2 .table-wrap}
::: {.caption}
######

Modulation of Surface Markers on BL36 Cells by rGal1
:::

  Surface Marker   Control 24 h   Treatment (% positive) rGal1 -- 24 h   Control 48 h   Treatment (% positive) rGal1-48 h
  ---------------- -------------- -------------------------------------- -------------- -----------------------------------
  CD71             98.00          94.90                                  93.00          45.00
  CD138            \<20           20.20                                  25.00          70.00

BL36 cells were exposed with 700 nM rGal1 for 1 and 2 days. Then cell surface antigens were analyzed by flow cytometry. Values below 20% were considered as negative.
:::

Discussion
==========

In 1994, Chiariotti et al. \[[@B16]\] first reported experimental data showing that the expression of Gal1 can be induced in cultured hepatoma-derived cells by treatment with AzaC. Interestingly, Gal1 and AzaC individually have been shown to affect similar cell processes in cancer cells, including differentiation, and growth inhibition \[[@B12]-[@B15],[@B27],[@B29],[@B46]-[@B51]\]. Available data are consistent with the suggestion that the expression of Gal1, accompanied by its secretion and its binding to cell surface receptors, could be involved in the AzaC observed effects in hematopoietic cells where Gal1 modulates differentiation or apoptosis. However, the mechanisms of these effects on hematopoietic cells are unclear yet.

In the present study, the effects of AzaC, on the cell phenotype, cell differentiation and cell death of BL36 cells were analyzed. The effects on cellular growth were time- and dose-dependent. Five μM AzaC caused significant inhibition on cellular growth, but the cell viability remained practically unchanged. AzaC increased the cell fraction in the G0/G1 phases, suggesting that AzaC inhibits cell division, which may be one of critical mechanisms of cell modulation by AzaC. AzaC-treated cells initially may become arrested at the G1 phase and then may either escape to the cycle arrest or die due to mechanisms leading to programmed cell death.

We also show that incubation of BL36 with AzaC induces expression of Gal1. AzaC is thought to exert its effects as a competitive inhibitor of cytosine methylation, resulting in the expression of silenced genes. The gene for Gal1 is one whose expression is possibly enhanced in this manner. In the results reported here, we found that Gal 1 was detected in cytosol after 120 h of treatment by 10 μM AzaC and then Gal1 was externalized and bound to cell surface receptors 24 h later. A key to understanding the extracellular biological functions of Gal1 is how its secretion appears to be regulated and re-directed during development and differentiation. Gal1 is likely released form vesicles close to the plasma membrane. On the basis of the data, we propose that the released Gal1 be immediately recruited to modulate cell activity. Gal1 may do this by interacting with and modulating cell receptors via its carbohydrate recognition domains because the Gal1-receptor interaction is abrogated by thiodigalactoside \[[@B29]\].

Others and we have previously reported that Gal1 binds to T and B lymphoblastoid cells \[[@B29],[@B52],[@B53]\]. Other studies have demonstrated that galectins are immunosuppressive, in animal models of autoimmune diseases \[[@B54]-[@B56]\]. Whereas the full role of Gal1 in modulating immune function is not yet understood, the increase in Gal1 expression by AzaC in BL cells suggests that Gal1 may play a role in the behaviour of normal leukocytes and of tumour cells.

What is the underlying mechanism? Although the regulatory machinery triggered by demethylating stimulus and resulting in phenotype modifications is not yet elucidated, it probably involves the stimulation of a signalling cascade that regulates cell proliferation and viability. A recently proposed model for such a cascade suggests the involvement of a cytoplasmic protein, AZ2 \[[@B57]\]. The amino-terminal part of the AZ2 protein is homologous to the previously reported TANK and I-TRAF, which participate in the signal transduction cascade from the TNF-receptor to the transcription factor NFkappaB. Demethylating stimulus may also modify a pathway activated by the membrane-anchored protein-tyro sine phosphatase CD45. Engagement of CD45 is known to regulate Src tyrosine kinases phosphorylation, phospholipase Cγ regulation, inositol phosphate production, diacylglycerol production, PKC activation, and calcium mobilisation \[[@B58]-[@B60]\].

Increased synthesis and secretion of Gal1 by the cell could account for part of the phenotypic alterations detected in AzaC treated cells. Gal1-induced dimerisation and/or segregation might inhibit the catalytic site in CD45, thereby blocking tyrosine phosphates activity. Because Gal1 binding to cell surface receptors results in tyrosine phosphorylation \[[@B29],[@B61]\], it may allow a kinase-dependent signal to be transduced. Several studies have linked Gal1 expression with growth inhibition \[[@B27]\] and cell death \[[@B26],[@B62]\]. However, the reports that some growth inhibitory agents did not induce Gal1 expression indicated that Gal1 expression is not dependent on the cell\'s growth state in general, through it may be involved in growth suppression \[[@B63]\]. Moreover, it is likely that Gal1 acts in a manner to regulate specific signal transduction processes that is determined by the cell type and by the state of cell differentiation. In this work, exogenous rGal1 added to BL cells inhibited cell growth. Moreover, Gal1 as well as AzaC induced an expression of the cell surface plasma cell antigen, CD138, a phenotypic marker that identify cells with plasmacytic differentiation \[[@B64]\]. This is consistent with the hypothesis that AzaC and Gal1 share similar signals for differentiation, however, since there was a significant difference in the expression of CD19 and CD23 after AzaC or Gal1 treatments it is likely that some pathways are specifically modified by AzaC. The mechanisms involved in these different pathways, important in clinical therapy, remain to be elucidated in the future. Ongoing studies are aimed at identifying as globally as possible the modifications resulting from AzaC treatment by using proteomics \[[@B65]\].

Acknowledgements
================

This work was supported, in part, by grants from the Ministère de l\'Education Nationale de la Recherche et de la Technologie (MENRT), and from the Ligue Française contre le Cancer (Comité de Seine Saint-Denis). FP was supported by ARC (Association pour la Recherche sur le Cancer).


Introduction
============

L-arginine (Arg), an essential amino acid, is required for normal growth of microbes, plants and animals. Deprivation of this amino acid from the culture medium or other sources of nutrition causes serious disturbances in cellular and organ function leading to total destruction. On the other hand, excessive doses of Arg also influence cell function, including cell death and cell proliferation. Substantial information has been obtained in the past decades on the role of Arg in tumour growth and in tumour therapy.

Effect of Arg deprivation and supplementation on tumour cell proliferation
==========================================================================

Arg, an essential amino acid, is required to maintain normal metabolism and proliferation of cells in culture \[[@B1]\]. Attempts to influence tumour cell proliferation by changes in amino acid balance were based on such observations. The role of the enzyme arginase, which decreases the amount of Arg, was thoroughly investigated in this respect and also used in the therapy of human tumours \[[@B2]\]. According to Umeda *et al.*\[[@B3]\], the proliferation of both HeLa cells in vitro and rat Novikoff hepatoma in vivo could be decreased by arginase, causing relative Arg deficiency. Otsuka \[[@B4]\] has shown that an enzyme, very similar to arginase inhibits DNA synthesis in normal rat liver. The proliferation promoting activity of L-Arg is also underscored by the fact, that Arg is converted by arginase to L-ornithine, which is the precursor of various polyamines essential for cell proliferation \[[@B5]\]. Tanaka *et al.*\[[@B6]\] have demonstrated the death of 3T3 cells after Arg deprivation. Wheatley *et al.*\[[@B7]-[@B10]\] analysed the effect of deprivation of eleven essential amino acids on several tumour cell lines and found that apoptotic-like cell death occurs as a consequence of this manipulation. The cell lines died considerably more quickly during Arg deprivation than in the absence of any other essential amino acids. Moreover, when co-cultures of normal and tumour cells were deprived of Arg the normal cells survived and the tumour cells died. According to these observations, Arg deprivation causes selective death of cultured malignant cells. Lamb and Wheatley \[[@B11]\] have also shown, that Arg deprivation most probably impairs the control of DNA synthesis at the G~1~ checkpoint, which normally inhibits its initiation of DNA synthesis under unfavourable conditions.

Arg imbalance was also produced by excess of Arg supplementation in the diet. Brittenden *et al.*\[[@B13]\] Suggested a possible therapeutic effect of Arg-rich diet in malignant disease, in combination with anti-cancer chemotherapy. Ogilvie *et al.*\[[@B14]\] found that excess Arg combined with doxorubicin chemotherapy extended disease-free interval and survival time of dogs with lymphoma. According to the studies of Hester and Fee \[[@B15]\] on squamous cell carcinoma in the CH3/KM mouse the mechanism of action of high amounts of Arg may be the stimulation of host immune surveillance. However, Robinson *et al.*\[[@B16]\] found that Morris hepatoma-bearing rats fed with Arg-rich diet did not show any alteration in tumour growth or cytokine production. The role of Arg in carcinogenesis has been challenged by the experiments of Weinberger *et al.*\[[@B17]\] who found that high doses of Arg glutamate decreased the carcinogenic activity of various acetamine-derivatives in rats.

Interesting data were reported on Arg-induced apoptosis of pancreatic acinar cells both in vitro and in vivo \[[@B18]\] providing a model of acute pancreatitis. The possible therapeutic use of Arg against pancreatic acinic cell carcinoma has not been examined yet.

Arg-rich hexapeptides were identified from peptide libraries that inhibit the interaction of vascular endothelial growth factor to its receptor. These hexapeptides inhibit the proliferation of human umbilical vein endothelial cells and also block the angiogenesis induced by vascular endothelial growth factor in vivo, in the chick chorioallantoic membrane and in the rabbit cornea. One of the hexapeptides blocks the growth and formation of metastases of HM7 human colon carcinoma cells in nude mice \[[@B19]\]. These results may serve as leads for development of anticancer drugs.

Arg imbalance was established in our early in vivo experiments \[[@B20]\]. High doses of L-Arg, D-Arg and DL-Arg (400--500--1000 mg/kg body weight intraperitoneally or orally) were administered to Wistar rats bearing subcutaneous Yoshida\'s sarcoma or to Swiss mice bearing subcutaneous Ehrlich carcinoma for 9--15 days (table [1](#T1){ref-type="table"}). D-Arg inhibited the growth of Yoshida\'s sarcoma significantly (50%, p \< 0.05), when applied in a daily dose of 500 mg/kg, orally. Intraperitoneal administration of the same dose to Ehrlich carcinoma bearing mice resulted in a 20%, statistically not significant, inhibition.

::: {#T1 .table-wrap}
::: {.caption}
######

Effect of D-Arg, L-Arg and DL-Arg on the growth of transplantable animal tumours
:::

  **Treatment**   **Tumour**     *Number of animals*   **Daily dose (mg/kg)**   **Duration of treatment (days)**   **Inhibition %**   *Remarks*
  --------------- -------------- --------------------- ------------------------ ---------------------------------- ------------------ ----------- -----------------------
  *D-Arg*^1^      Yoshida        6                     6                        500 p.o.                           12                 50          p \< 0.05
  D-Arg ^1^       Ehrlich s.c.   10                    10                       500 i.p.                           10                 20          p \< 0.1
  L-Arg^3^        Yoshida        10                    10                       400 i.p.                           10                 -40         Enhancement p \< 0.05
  L-Arg^3^        Yoshida        10                    10                       400 p.o.                           9                  -30         Enhancement p \< 0.1
  L-Arg^2^        Ehrlich s.c.   10                    10                       500 i.p.                           12                 -40         Enhancement p \< 0.05
  DL-Arg^3^       Ehrlich s.c.   10                    10                       500 i.p.                           12                 \-          \-

^1^Serva (USA), ^2^Ajinomoto (Japan), ^3^Reanal (Hungary) Animals were sacrificed 24 hours after the last treatment and tumour weight was measured. Inhibition or enhancement is given as per cent of the control tumours. Abbreviations:i. p. -- intraperitoneal; p. o. -- peroral; s. c. -- subcutaneous; Arg -- arginine
:::

L-Arg, however, enhanced the growth of Yoshida\'s sarcoma, when given intraperitoneally or orally in a dose of 400 mg/kg. The same tendency, namely significant (40%) enhancement was seen after intraperitoneal treatment (400 mg/kg) of Ehrlich carcinoma bearing mice.

Intraperitoneal application of 500 mg/kg DL-Arg to mice, inoculated with Ehrlich carcinoma had neither inhibitory nor enhancing effect on tumour growth. Various pathways of metabolism of this amino acid may explain the mode of action of Arg imbalance. Among these methylation and hydroxymethylation appear to be of special importance.

Effect of methylated, hydroxy and hydroxymethylated Arg on cell death and proliferation
=======================================================================================

Arg is a highly reactive compound, both as a free amino acid and as a constituent of a protein. The structure of proteins can be altered by specific enzymatic modification of the side chains. One of these protein-modifying reactions is methylation, resulting in the addition of methyl groups to the guanidine residues of Arg. Methylated Arg also occur in free form, possibly resulting from enzymatic hydrolysis of methylated proteins in vivo (fig. 1).

Mono-di- and trimethyl Arg, hydroxymethyl Arg, N-omega-hydroxy L-Arg, N-nitro-L-Arg methyl ester, Nitro-Arg were studied regarding the possible effect on cell death and cell proliferation of these compounds.

Tyihák *et al.*\[[@B21]\] demonstrated that monomethyl Arg and dimethyl-Arg inhibit significantly the growth of tobacco tissue cultures in concentrations of 10--100 ppm in agar nutrient medium, NG-methylated Arg added to agar-medium also significantly inhibited the growth of the roots of lettuce seedlings. Further investigations along this line carried out by Szende *et al.*\[[@B22]\] have shown that NG-hydroxymethyl Arg inhibited dose-dependently and significantly the proliferation of HT-29 human colon carcinoma cells, P-388 mouse lymphoma cells and PC-3 human prostate carcinoma as well as K-562 human erythroleukaemia cells in culture. The cells of the treated cultures showed morphological signs of apoptosis in a high percentage. In our in vivo experiments \[[@B20]\] hydroxymethylated Arg was administered to Swiss mice inoculated with Ehrlich ascites tumour. The daily dose of NG-hydroxymethyl Arg was 400 mg/kg intraperitoneally, based on previous acute toxicity studies. After 10 days of NG-hydroxymethylated Arg treatment complete inhibition of the growth of Ehrlich ascites tumour was observed.

C57B1 mice, inoculated with Lewis lung tumour intramuscularly and made tumour free by amputation of the tumorous leg 10 days after tumour transplantation, were treated for seven days with 400 mg/kg NG-hydroxymethyl Arg, daily, intraperitoneally. The treatment started 24 hours after amputation. The animals were sacrificed on the 8^th^ day after starting the treatment. Lung metastasis number and volume were determined. The average metastasis number in the treated animals was 27, in the controls 54. The average volume of the lung nodules was 34 mm^3^ in the treated and 50 in the control mice.

The anti-proliferative and apoptosis-inducing effect of Arg-derivatives was confirmed by the studies of Singh *et al.*\[[@B5]\] who found that N-omega-hydroxy-L-Arg inhibited the proliferation of the high-arginase-expressing MDH-MB-468 cells and induced apoptosis after 48 hours. It has also been shown by Washo-Stultz *et al.*\[[@B23]\] that N-nitro-Arg methyl ester sensitised cells to apoptosis induced by sodium deoxycholate.

L-Arg, nitro-Arg and methyl-Arg have been found to induce increase in cytosolic Ca concentration in cultured NIT-1 cells \[[@B24]\], leading to depolarisation of the plasma membrane potential, a phenomenon common during the process of apoptosis.

NG-methyl-L-Arg \[[@B25]\] N-nitro-L-Arg methyl ester \[[@B23]\], N-hydroxy-L-Arg \[[@B5]\] and L-NG-methyl-Arg \[[@B26]\] all proved to be inhibitors of nitric oxide synthase. Nitric oxide synthase converts L-Arg to produce NO, which \"Janus-faced\" compound certainly may influence both cell proliferation and cell death.

Role of formaldehyde in the mechanism of action of methylated arginines
=======================================================================

Another possible mode of action of methylated and hydroxymethylated Arg can be deduced from the fact that these molecules are formaldehyde generators. It has been demonstrated by Hullán *et al.*\[[@B27]\] that NG-hydroxymethyl Arg as a biomolecule is one of the compounds that are responsible for the endogenous formaldehyde level. The guanidine group of L-Arg can bind one, two or three molecules of CH~2~O and in the reaction mono-, di- and trihydroxymethylated Arg derivatives are formed. This process is catalysed by the enzyme transmethylase \[[@B28]\]. The hydroxymethylated derivatives of Arg are relatively stable compounds. Arg is suitable to carry the endogenous CH~2~O in form of hydroxymethyl group in biological systems. The hydroxymethyl groups are attached to the guanidine group by reversible bindings \[[@B29]\]. Although little is known about the demethylation of NG-methylated Arg, NG-hydroxymethyl-L-Arg generates a direct CH~2~O-yielding activity, which may be responsible for its apoptotic effect \[[@B22],[@B30]\]. It has also been shown in our recent experiments \[[@B31]\] that the administration of analytically pure formaldehyde to cell cultures causes dose dependently apoptosis (1--10 μg/ml) or stimulation of DNA synthesis and cell proliferation (0.1--0.01 μg/ml). The calculated quantity of formaldehyde released by demethylation processes from hydroxymethyl Arg is in the above mentioned range and the formaldehyde-mediated biological action of this compound has to be taken into consideration.

Lysine-arginine antagonism
==========================

The lysine-arginine antagonism widely shown in nature is also represented in apoptosis resistant cell lines that contain A-to G alteration is the death domain, encoding L-arginine instead of L-lysine in codon 441 \[[@B32]\].

Arg 72-p53 genotype and cancer
==============================

Over-representation of the homozygous Arg 72-p53 genotype in cervical carcinoma patients has been reported \[[@B33]\]. However, Tachezy *et al.*\[[@B34]\] did not find increased risk for human papilloma virus-associated cervical tumour development associated with Arg 72-p53.

Conclusions
===========

Although data in the literature are pointing to the anti proliferative effect of both Arg depletion and supplementation, Arg proved to be essential for tumour cell growth. This observation raises the question, whether decreasing the concentration of Arg in nutrients and consequently in blood serum or the administration of D-arginine may lead to retardation of tumour growth in humans, too. Arg, because of its strong basic guanidine group, plays an important role in molecular interactions in biological systems, such as interaction between Arg and formaldehyde, both of which are normal components of cells and biological fluids. As a result, hydroxymethyl derivatives of Arg are formed. These compounds may be the source of formaldehyde generation. Arg may be considered a formaldehyde capturer, carrier and generator molecule. These functions may also play role in the biological activity of Arg and its methylated and hydroxymethylated derivatives. An interesting therapeutic possibility worthy of further investigation may be the administration of methylated and hydroxymethylated Arg in order to induce tumour cell death or to prevent tumour cell proliferation.


Background
==========

Arterial complications in Behcet\'s disease are rarely seen and usually involve great arteries like aorta and iliac arteries, and may lead to the perforation of the arterial walls and to the development of the aneurysms and their rupture \[[@B1]\]. Due to weakness and fragility of the aortic wall and arteritis open surgery has some complications like the development pseudoaneurysms. In this case report, an infrarenal huge saccular abdominal aneurysm extending to the left inguinal region in a 32 years old male Behcet patient that was first seen in spinal CT examination performed due to extensive back pain and consequently had a successful stent graft placement is presented.

Case presentation
=================

A 32 years old male patient with intense back pain admitted to the emergency room. From his history it was learnt that he had Behcet disease and being followed by another institution. Direct roentgenograms revealed a smooth contoured density increase on left side of vertebral column (Figure [1](#F1){ref-type="fig"}). Lumbar vertebral CT scan revealed significant destruction on the anteriolateral parts of vertebral corpuses between L2 and L4, more prominent on L3 and hypodense area noted. Then dynamic abdominal CT was performed which showed an infrarenal, 10 × 10 × 10 cm saccular aneurysm originating form a 3.5 × 1 cm defect on the left posterolateral aspect of abdominal aorta, with thrombus inside (Figure [2A, 2B](#F2){ref-type="fig"}). Due to the risk of pseudoaneurysm development digital subtraction angiography (DSA) was not performed and CT angiography (CTA) and 3D reconstructions were made (Figure [3A, 3B, 3C, 3D, 3E](#F3){ref-type="fig"}). CT examinations were performed with GE ProSpeed Helical scanner and for the reformat images and 3D reconstructions Advantage Windows version 2.0 Voxtool software on a separate workstation was used. The patient was not considered an ideal candidate for open surgery and endovascular stent graft placement was planned. All the measurements made by using abdominal CT and CTA images that revealed favorable lengths and diameters for endovascular treatment. A nitinol, polyster covered 14 × 70 mm stent graft (Stenford Grouppe Valendons fa Nanterre, France), beginning from 1.5 cm proximal and 1.5 cm distal to the aortic defect, ordered. Intervention took place in the operating room under spinal epidural anesthesia with the guidance of Schimadzu C-Arm fluoroscopy equipment that had no DSA function. Systemic iv 5000 U heparin was administered. Right common femoral artery was exposed and an arteriotomy was made. A 12 F (75 cm length) introducer was advanced and stent graft was deployed 1.5 cm below the renal arteries. During deployment systemic arterial pressure dropped to 50 mmHg. Intraprocedural control conventional angiography showed the patency of renal arteries and proper position of the deployed stent graft and entrapped contrast material inside the aneurysm sac (Figure [4A, 4B](#F4){ref-type="fig"}). Arteriotomy site was closed by primary sutures to prevent pseudoaneurysm development and wrapped by woven collagen covered Dacron graft (Hemashield, Meadox Medicals, Oakland, NJ). There was no intervention related intra or post procedural complications. Control abdominal CT examination and 3D reconstructions revealed complete sealing of the aneurysm sac by the stent graft, patency of the renal arteries and visceral arteries and no endoleak was noted (Figure [5A, 5B, 5C, 5D, 5E](#F5){ref-type="fig"}). No ischemic complications in the lower extremities were encountered. Patient is discharged with steroid therapy and is being followed up serial abdominal CT. After 18 months there were no complications (Figure [6A, 6B](#F6){ref-type="fig"}).

![Direct roentgenogram of the abdomen shows smooth contoured density increase on left side of vertebral column.](1471-2342-2-1-1){#F1}

![A, B. Abdominal helical CT images show prominent destruction in the left anterolateral portion of the lumbar vertebra corpus and 10 × 10 × 15 cm saccular aneurysm originating from a 3.5 × 1 cm defect in the left posterolateral aspect of the abdominal aorta and thrombus inside. The vertebral destruction was probably due to pulsation of the aneurysm.](1471-2342-2-1-2){#F2}

![A, B, C, D, E. Sagittal reformat CT image (A), surface shaded display (SSD) 3D images (B, C) and maximum intensity projection (MIP) multiplanar volume reformat (MPVR) images (D, E) show neck of the aneurysm, anterior displacement of the aorta and patency of the iliac bifurcation and iliac arteries.](1471-2342-2-1-3){#F3}

![A, B. C-arm fluoroscopy image obtained during the procedure in the operating room show the patency of renal arteries (A) and deployed stent graft (B). Arrowhead points to the contrast material entrapped inside the aneurysm sac.](1471-2342-2-1-4){#F4}

![A, B, C, D, E. Abdominal helical CT (A), sagittal reformat CT (B) and SSD images (C) show non-filling of the aneurysm sac and complete sealing of the defect. Abdominal CT images shows right (D) and left (E) patent renal arteries.](1471-2342-2-1-5){#F5}

![A, B. Control abdominal CT image after 18 months shows a decrease in the size of the aneurysm sac and patency of the stent graft. No endoleak was noted.](1471-2342-2-1-6){#F6}

Discussion
==========

Behcet disease is first described by Hulusi Behcet, a Turkish dermatologist, in 1937 \[[@B2]\]. It is a multisystemic inflammatory disease that classically causing oral and genital ulcers, and ocular inflammation. It can also affect vascular system. Since it can affect both arteries and veins of all sizes, Behcet vasculopathy is different from other vasculitides \[[@B3]\].

There is no pathognomonic laboratory test or histologic finding specific to Behcet disease. Thus the diagnosis is based on clinical criteria. Various criteria were proposed (like Mason and Barnes, The Behcet\'s disease Research Committee of Japan, O\'Duffy and Goldstein, International Study Group (ISG) etc). The most commonly used criteria of ISG \[[@B4]\] require recurrent oral ulceration plus at least two of the following: recurrent genital ulcerations, eye lesions (like uveitis etc), skin lesions (erythema nodosum, folliculitis etc), positive pathergy test. ISG considered subcutaneous thrombophlebitis, deep vein thrombosis and arterial aneurysms as a criterion. Although these have high specifity for Behcet disease, they are not accepted as criteria due to their low sensitivity.

The etiology of Behcet disease is still not certain but systemic vasculitis has an important role. Perivascular lymphocytic and plasma cell infiltration, endothelial cell proliferation and swelling, disruption of elastic lamina, degeneration of the tunica media and vasculitis of the small vessels in vasa vasorum are among the histologic features of venous and arterial lesions \[[@B5],[@B6]\]

Behcet vasculopathy includes arterial and venous thrombosis formations, subcutaneous thrombophlebitis, venous collateralizations, pulmonary artery aneurysm, arterial aneurysm and occlusions \[[@B6]\] Arterial aneurysms are more common than arterial thrombosis. Although aorta is the most common site of involvement pulmonary, femoral, subclavian, popliteal and carotid arteries also could be affected. Histologic examinations of the arterial lesions show vasculitis in the vasa vasorum together with thickening in the media and separation of the elastic fibers, which in turn causes development of aneurysms. \[[@B6]-[@B8]\].

Endovascular grafts provide an important alternative to high-risk patients with more invasive surgical procedures. Patients who had endovascular stent graft placement had shorter operating room and hospital stay and less blood loss than open surgery. From the economical stand point the cost of stent graft placement is much higher than open surgery in our country.

Although numerous improvements in catheter, stent and graft technology have been made, there are still some limitations like the size of delivery systems, endoleaks, position of the renal arteries etc. The success rate of endovascular stent grafting is high (90% in low risk patients, 80% in moderate to high risk patients). Short-term mortality (within the 30 days following the procedure) is reported to be 2.5% in low risk group, 8% in high-risk group. Although there is disagreement in reported mortality rates for open surgery \[[@B9]\], there are reports of mortality rates of between 1.6 % \[[@B10]\] to 7,6 % \[[@B11],[@B12]\]. As it is common for all new or minimally invasive techniques, there is a learning curve for endovascular stent grafting that affects both the results and costs. Thus for the best results efficiency and experience are needed.

One of the crucial points of endovascular treatment of aneurysms is accurate and precise measurement of aneurysm. Although CT is highly effective, most of the vascular radiologists still would like to have preprocedural angiography. But in Behcet disease vessel wall vulnerability is extremely high and even minor lesions resulting from diagnostic procedures (e.g. intraarterial angiography etc.) could lead to the formation of aneurysms at the puncture site \[[@B13]\]. Due to the fragility of the aneurysm inherent in Behcet patients and the size of aneurysm, we did not perform diagnostic angiography. All measurements based on CT images and 3D reconstructions helped us to better understand and characterize the neck, dimensions and relations of the aneurysm to other arteries.

Endovascular radiological intervention in the treatment of abdominal aortic aneurysm complications of young Behcet patients, in whom surgery is not suitable and intervention is necessary, could be an alternative treatment modality even without performing preprocedural conventional angiography or DSA using the advantages of helical CT and 3D reconstruction techniques.

Competing interests
===================

none declared

Authors\' contributions
=======================

Author 1, RK, conceived of the study, carried out the imaging work-up and the endovascular procedure, and drafted the manuscript.

Author 2, OG, carried out the surgical part and participated in drafting the manuscript

Author 3, AA, participated in the imaging work-up and en do vascular procedure.

Author 4, RT, participated in the surgical part, and coordination.

Author 5, TB, participated in the imaging work-up and coordination.

All authors read and approved the final manuscript.

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>

Acknowledgements
================

Written consent was obtained from the patient for publication of the patient\'s details.. The manuscript has been presented in the TURK RAD, 17th National Radiology Congress (27--31 September 2000, Istanbul, Turkey) as a poster presentation.


Background
==========

Diabetes mellitus constitutes a major Australian public health problem. It has been identified as one of our national health priority areas\[[@B1]\], and recent data suggests that up to 7.5% of the Australian adult population now suffers from this condition. After 20 years of diabetes most patients can be expected to develop diabetic retinopathy\[[@B3]\] which, if undetected, is likely to cause significant visual loss or blindness. A recent Australian survey recorded almost 1/3 of patients with self-reported diabetes having diabetic retinopathy.\[[@B4]\] However, if detected early by appropriate retinal examination, followed and treated by laser photocoagulation, most patients can be saved from major visual loss. \[[@B5]-[@B7]\]

Our National Health and Medical Research Council (NHMRC) Guidelines on the management of diabetic retinopathy, recommend regular ocular review to detect, treat and minimize such visual morbidity. \[[@B8]\] Recent Australian studies, however, have documented only a relatively small proportion of Australians with diabetes receiving such sight-saving examinations. The Melbourne Visual Impairment Project, a large population-based epidemiological study of 4,744 Victorians with diabetes, found that only half the sample surveyed reported a retinal examination within the last 2 years. Worryingly, participants with diabetic retinopathy were no more likely to have had an examination than those without (52.9% v 49.3% p = 0.66)\[[@B3]\]. The National Divisions Diabetes Program, a survey of the treatment of diabetes in 4,359 patients across 7 Divisions of General Practice, recorded 49.6% of patients to have had an appropriate retinal examination over the 24 month reporting period. \[[@B9]\]

A number of strategies have been implemented to increase the number of Australians with diabetes accessing regular eye checks. Remote clinics with annual \"fly-in\" ophthalmologists operate in a number of Australian states. Mobile retinal cameras, operated by state health departments or Divisions of General Practice, have recently improved community access to screening,\[[@B10]\] but are expensive to purchase and operate. Australian optometrists are offering their services in the area of diabetic retinopathy screening. However, little attention to date has focused around the use of appropriately-trained general practitioners in offering effective and accessible screening for diabetic retinopathy. We aimed to determine whether general practitioners could be trained to identify the important features of diabetic retinopathy in a short training time and with an acceptable degree of sensitivity and specificity.

Method
======

Seventeen GPs from the Bayside Division of General Practice self-selected to participate in a Divisional Eye Upskilling Project, funded by the Department of Health and Human Services. As part of this program, the University of Queensland Departments of Ophthalmology and General Practice were asked to design a brief \'hands-on\' educational intervention to assist GP skill development in the ocular assessment and management of diabetes mellitus and primary open angle glaucoma. The complete program and its evaluation has been documented in a previous paper .\[[@B11]\] Details of the diabetic retinopathy training intervention are displayed in Table [1](#T1){ref-type="table"}.

::: {#T1 .table-wrap}
::: {.caption}
######

Diabetes segment of the GP Eye Upskilling Program
:::

  Workshop 1                                       Workshop 2
  ------------ ----------------------------------- ------------ ----- -------------------------------------------------- ---------
  1\.          Overview diabetic eye disease       30 mins      1\.   Patient fundoscopy clinic                          30 mins
  2\.          Fundoscopy video                    15 mins      2\.   Question and answer session                        15 mins
  3\.          Fundoscopy practice                 30 mins      3\.   Use of skills in the general practice time frame   15 mins
  4\.          Slide review session                15 mins      4\.   Slide review                                       15 mins
  5\.          Patient fundoscopy clinic session   35 mins
:::

All sessions were highly interactive and used patients for skill demonstration wherever possible. These sessions were conducted within the Professorial Eye Unit, Princess Alexandra Hospital, using patients with good clinical signs. Doctors were urged to practice and refine skills in their own practices between workshops, and to comment on barriers and supports to the use of the skills in their clinical practice.

All GPs underwent a comprehensive practical assessment of their eye skills prior to undertaking the intervention, and completed an identical assessment at the conclusion (pre and post test). The pre and post evaluation relevant to the diabetes retinopathy skills component consisted of eight (8) two-minute patient fundal examinations conducted a month prior and a month following the skills program. Four (4) of the patients involved in this assessment suffered from diabetic retinopathy, and four (4) had normal fundi.. The gold standard used to determine the presence or absence of diabetic retinopathy was the agreed clinical assessment by two academic ophthalmologists. All four diabetic (4) patients had background diabetic retinopathy. Every attempt was made to make pre and post evaluation assessments for the diabetes patients identical. However, one of the four diabetic patients participating in the pre-test, was unable to return for the final evaluation session. He was replaced, as closely as possible, by a patient with similar fundal appearance. The four patients with normal fundi were recruited from the ward and outpatient departments on the day of assessment. One \"normal\" failed to attend the post-test. The post test was in all other respects conducted identically to the pre test. GPs were not informed of the retinopathy status of the patients used in the pre-evaluation testing and these patients were separated from clinical teaching sessions during the educational intervention.

All patients were unknown to participating GPs. The University of Queensland ophthalmology staff supervised all training sessions and both assessments. Ethical approval to conduct the study was sought and received from the Princess Alexandra Hospital Ethics Committee.

Statistical method
------------------

Participant performance on the patient fundal assessment, pre and post educational intervention, was used to determine sensitivity and specificity of screening for diabetic retinopathy. Sensitivity was calculated as the ratio of patient assessments correctly identified by the GPs as diabetic, to the total number of diabetic retinopathy positive cases (four subjects in total in both pre and post tests). Specificity was calculated as a ratio of the number of normal cases correctly identified over the total number of normals participating in both tests (four subjects in the pre-test, and three subjects in the post-test). \[[@B12]\]

For each GP, sensitivity pre and post educational intervention was classified \'not satisfactory\' if sensitivity was less than 60% and classified \'satisfactory\' if sensitivity was equal to or greater than 60%. For each GP, specificity pre and post educational intervention was classified \'not satisfactory\' if specificity was less than 60% and classified \'satisfactory\' if specificity was equal to or greater than 60%. McNemar\'s Test was then used to determine if there was an association between Sensitivity Classification and educational intervention. This test was used because it accounts for the paired nature of the data. That is, the same GPs were evaluated Pre- and Post-workshop. Statistical significance is quoted at the conventional p \< 0.05 level. All hypothesis testing was based on two-tailed hypotheses. Due to small sample sizes exact significance levels were used.

Results
=======

Sensitivity
-----------

Pre and post intervention sensitivity results are displayed as percentages in Table [3](#T3){ref-type="table"}. Ten GPs (59%) achieved a screening sensitivity of 25% or less in the pre test, three (18%) scored 50%, two (11.5%) scored 75%, and two (11.5%) recorded 100% sensitivity. In the post test, all seventeen GPs achieved between 50 and 100% sensitivity (1 GP achieved 50% sensitivity, 11 GPs (65%) achieved 75% sensitivity, and 5 GPs (29%) achieved 100% sensitivity).

::: {#T3 .table-wrap}
::: {.caption}
######

Sensitivity of GP screening for Diabetic Retinopathy (n = 17)
:::

        Pre-workshop   Post-workshop
  ----- -------------- ---------------
  0     4              0
  25    6              0
  50    3              1
  75    2              11
  100   2              5
:::

The McNemar test (Table [4](#T4){ref-type="table"}) was statistically significant and there was an association between sensitivity classification and educational invention (p = 0.001). There was a significant increase in the number GPs with \'satisfactory\' sensitivity from Pre- to Post-workshop.

::: {#T4 .table-wrap}
::: {.caption}
######

Specificity of GP screening for Diabetic Retinopathy (n = 17)
:::

  Pre-workshop   Post-workshop
  -------------- --------------- ------ ---
  0              3               0      1
  25             6
  50             4               33.3   3
  75             4               66.6   6
  100            0               100    7
:::

Specificity
-----------

Specificity results, pre and post intervention, are displayed in Table [5](#T5){ref-type="table"}. As one of our \"normals\" failed to appear in the post test, the number of subjects used in the post assessment dropped from four to three, hence the specificities are expressed in increments of 25% for the pre test compared with increments of 33% for the post test. In the pre test, nine GPs (54%) recorded a screening specificity of less than 50%, four (23%) a specificity of 50% and four (23%) a specificity of 75%. In the post test, only four GPs (23%) recorded a specificity of less than 50%, six (35%) a specificity of 66%, and seven (41%) 100% specificity. No GP recorded 100% specificity in the pre test, whilst 7 (41%) recorded 100% screening specificity following the intervention.

::: {#T5 .table-wrap}
::: {.caption}
######

Sensitivity Classification of GP screening for Diabetic Retinopathy by Pre-and Post-workshop
:::

                                     Pre-workshop   Total
  --------------- ------------------ -------------- ------- ----
  Post-workshop   not satisfactory   1              0       1
                  satisfactory       11             5       16

  Total                              12             5       17
:::

The McNemar test (Table [6](#T6){ref-type="table"}) was statistically significant and there was an association between specificity classification and educational invention (p = 0.001). There was a significant increase in the number GPs with \'satisfactory\' specificity from Pre- to Post-workshop.

::: {#T6 .table-wrap}
::: {.caption}
######

Specificity Classification of GP screening for Diabetic Retinopathy by Pre-and Post-workshop
:::

                                     Pre-workshop   Total
  --------------- ------------------ -------------- ------- ----
  Post-workshop   not satisfactory   3              1       4
                  satisfactory       10             3       13

  Total                              13             4       17
:::

The NH&MRC publication \"Management of Diabetic Retinopathy: a guide for general practitioners\' \[[@B8]\] nominates that a \"60% detection rate of early Diabetic Retinopathy (DR) may be sufficient for a successful screening campaign, so long as diabetic patients deemed to not have DR are screened at least every two years.\" In this study, 24% of GPs met this criterion prior to the workshop, and 94% met it following the intervention.

Discussion
==========

The early identification of diabetic retinopathy is crucial, as effective therapy is now available for the maintenance of vision in most patients. However, the absence of a systematic approach to such identification for Australians with diabetes limits our ability to provide optimal visual outcomes. The limited number of ophthalmologists available in the community restricts their role to the confirmation of diabetic retinopathy, grading, and eventual treatment. The use of a mobile fundus camera and central reading of all photos remains a potential method of identification of retinopathy, although as yet this has not been widely embraced in any single large community (outside remote areas).

GP screening is rarely suggested as an effective means of improving the early detection of diabetic retinopathy in Australia, yet it offers a number of clear benefits. General practitioners have almost universal access to Australian adults with diabetes, as GPs provide the bulk of adult diabetes diagnosis and care. As well as managing glucose control, medication, and lifestyle issues, GPs perform regular checks for peripheral neuropathy, nephropathy and macro vascular disease. An assessment for retinopathy could easily be included as part of the existing annual diabetes assessment (as nominated under the latest RACGP / DA Guidelines). This would negate the need to purchase and move expensive machinery, or arrange separate appointment times and assessments, and would be cost-effective for both the patient and community. A general practice screening model also offers the potential to opportunistically \"catch\" unscreened diabetics presenting to the general practitioner in a variety of other situations. General practitioners have a pre-established referral link with local ophthalmologists if early diabetic retinopathy is suspected. In most Australian states, this relationship has increasingly included an educational element via the RACGP / RACO National GP Eye Skills Program.

A GP screening model thus has the potential to provide a widely-available, holistic and extremely cost effective screening service on a population basis -- providing general practitioner skills are adequate for the identification process.

Previous work with general practitioners in this area suggested to us that GP reluctance to undertake retinal screening was due to concerns about the time taken for the examination; a long-standing and unfounded fear of the consequences of pupillary dilatation; and a lack of confidence in undertaking funduscopy with a direct ophthalmoscope. McCarty et al (13) identify lack of dilating drops in the practice, lack of confidence in detecting changes, concern re time taken and the fear of precipitation of angle-closure glaucoma as major barriers to GPs performing dilated ophthalmoscopy with their patients. Strategies to address all of these issues and perceptions were addressed as part of the \"\_How will this change my practice?\" module in our educational intervention (Table [2](#T2){ref-type="table"}). Follow up of a separate group of GPs taking this intervention as part of the national RACGP / RACO GP Eye Skills Workshop has demonstrated a sustained change in reported practice in this area \[[@B14]\].

We piloted a brief interactive training intervention to address these issues. Our GP intervention group reviewed and accepted recent data on the rarity of acute glaucoma following pupillary dilitation with short-acting mydriatics, and rapidly acquired effective skills in dilated diabetic fundal assessment. A focus of the training was the application of skills in each individual practitioner\'s work environment, and the practicing of skills between sessions in the general practice setting. This pilot aimed to assess the efficacy of this teaching program in raising the skills of those involved to the level of the current NHMRC guidelines for diabetic retinopathy screening. However, until patients are screened more regularly, the detection rate for diabetic retinopathy may need concomitantly to be higher than the 60% nominated in this guideline.

::: {#T2 .table-wrap}
::: {.caption}
######

Workshop Modules (National RACGP / RACO GP Eye Skills Workshop)
:::

  ----------- -----------------------------------------
  Module 1    Vision Testing And Ophthalmic Screening
  Module 2    Concepts And Assessment For Glaucoma
  Module 3    The Slit Lamp
  Module 4    Opthalmoscopy
  Module 5    Incision Of Chalazion
  Module 6    Removal Of Corneal Foreign Bodies
  Module 7    Suture Repair Of Entropion
  Module 8    Ocular First Aid
  Module 9    The Six Point Eye Examination
  Module 10   How Will This Change My Practice?
  Module 11   Patient Assessment Clinic (Optional)
  ----------- -----------------------------------------
:::

Following training, 94 % of the general practice study group reached this level within a two minute patient examination (both eyes). In addition, the specificity of the identification was such that it would not provide a gross referral overload to ophthalmologists for more accurate assessment and treatment. The time and effort required could be easily integrated into an annual review for patients with diabetes. The Royal Australian Colleges of General Practice and Ophthalmology have implemented this workshop across Queensland in 2000.

With appropriate training, motivation and support, GPs can make a major contribution in the area of effective screening for diabetic retinopathy. Clinicians, educators, bureaucrats and economists within our health system should be aware of this in framing future strategies in the area of diabetes.

Competing interests
===================

None declared

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>

Acknowledgements
================

The authors would like to thank the project manager Manjula Reedy, and the Bayside GPs for their contributions.


Background
==========

Collagen in the intervertebral disc, tendons, ligaments, heart valve leaflets, intestine and cornea displays a wavy collagen pattern termed \"crimping\". This crimp morphology contributes an important component to the mechanical responsiveness of these tissues \[[@B1]-[@B4]\]. When these tissues experience loading, the collagen crimp architecture is gradually straightened; this makes possible the slight elongation of collagen at the loaded site.

The biomechanical behavior of the disc is closely linked to this specialized crimp morphology (Figure [1](#F1){ref-type="fig"}) \[[@B2],[@B3]\]. The collagen fibers which form the annulus possess the same planar crimped geometry as is seen in the tendon. In the disc, however, crimp parameters vary according to the radial distance through the annulus. The lamellar structure of the annulus consists of continuous layers of collagen fibers that encircle the nucleus pulposus. In successive lamellar layers, the fiber orientation changes with respect to the spinal column axis. From the outer annulus inward, the interlamellar angle decreases linearly. Within each lamellar bundle, the collagen fibers are aligned in parallel arrays, and the fibers display a planar crimped waveform with all fibers in register. A gradient of crimp angle also is present in the disc, with crimp angle increasing and the crimp period decreasing from the periphery of the disc inward.

![Diagrammatic illustration of the organization of the intervertebral disc with attention to lamellar structure in the annulus and crimping in the collagen fibers. (Reproduced with permission \[[@B3]\].](1471-2474-3-9-1){#F1}

This complex and sophisticated architectural organization of lamellar disc structure serves to meet the unique biomechanical needs of the healthy disc. Although it is an important aspect of disc structure and function, this topic, which addresses the interface of microscopic anatomy and biomechanical function, has not been studied with specific reference to changes in the disc architecture during aging and degeneration. In the present report we suggest that the important stretch response of collagen made possible by collagen crimping is altered by morphologic changes in disc architecture during aging and degeneration. This alteration in the crimping behavior of collagen may thus contribute to the underlying tissue changes which culminate in fissures and annual tears in the degenerating disc.

Methods
=======

Control (normal) donor disc specimens were obtained from disc surgeries and the NCI Cooperative Human Tissue Network (CHTN). Studies were approved by the human subjects Institutional Review Board. Disc tissue studied here was from the annulus. Patient specimens were obtained from surgical procedures performed on individuals with herniated discs. Electron microscopy was used in the study of a total of 78 subjects (29 were from control donors \[aged newborn to 79 years\], and 49 were from surgical specimens from patients \[aged 16 to 77 years\] undergoing disc surgery). Light microscopic studies utilized the annulus from 40 control donors and 53 surgical specimens. The majority of the specimens studied here were from anterior sites of lumbar discs.

Surgical specimens were transported to the lab within 30 minutes of surgical removal in sterile modified minimal essential medium with Earle\'s salts (MEM, Gibco, Grand Island, NY) with 1% (v/v) L-glutamine (Irvine Scientific, Santa Ana, CA), 1% (v/v) nonessential amino acids (Irvine Scientific), and 1% (v/v) penicillin-streptomycin (Irvine Scientific). CHTN specimens were shipped overnight in sterile MEM. Upon receipt into the lab, specimens from control donors were carefully dissected to obtain specimens from the outer annulus, inner annulus and nucleus.

For electron microscopy, specimens were minced and fixed in Karnovsky\'s fixative, post-fixed with osmium tetroxide supplemented with 0.1% ruthenium red to enhance proteoglycan visualization in the extracellular matrix \[[@B5],[@B6]\], embedded in Spurr resin, thin sectioned and grid stained with uranyl acetate and lead citrate. In order to obtain representative specimens to examine with electron microscopy, 4--6 blocks of tissue were trimmed, thick-sectioned and examined with light microscopy and 1--2 blocks (in which the plane of sectioning was parallel to the long axis of the collagen fibrils) selected for thin sectioning. Sections were viewed on a Phillips CM10 transmission electron microscope.

For light microscopy, specimens were fixed in 10% neutral buffered formalin, embedded in paraffin or glycol methacrylate, and stained with picrosirius red-alcian blue, Masson trichrome, or Goldner\'s stain. Three-dimensional intensity graphical presentation were obtained using BioScan^®^ OPTIMAS™ (BioScan, Edmonds, WA). Morphologic images were captured with a CoolCam 2000 video system and frame grabber (Cool Camera Company, Decatur, GA) and prints were prepared with a Sony Mavigraph color video printer (Sony Electronics, San Jose, CA).

Results
=======

Opportune sectioning planes through the annulus reveal collagen crimping at both the ultrastructural and light microscopic level. Figure [2A](#F2){ref-type="fig"} illustrates the normal annular morphology of a healthy young intervertebral disc. Disc cells are flattened and lie in a modest lacunar space within collagen lamellar bundles. Prominent wavy expanses of collagen (collagen crimping) are present. Aging and degenerating disc matrix, however, shows striking alterations in tissue architecture. Changes include the presence of layers of unusual extracellular matrix encircling disc cells and interterritorial matrix alterations which extend into the surrounding lamellar structure (Figure [2B](#F2){ref-type="fig"}). In these regions collagen fibers are fewer in number and fibers are separated from each other reducing collagen crimping areas. Sites of more advanced degeneration can contain areas where there is prominent focal loss of matrix (Figure [2C](#F2){ref-type="fig"}). Only sparse regions of collagen remain in such sites.

![Figs. [2A](#F2){ref-type="fig"} and [2B](#F2){ref-type="fig"}: Photomicrographs of light microscopic features of the annulus in a disc from a three month old infant (A) and a control disc from a 68 year old female (B and C). Arrows in A mark regions of prominent collagen crimping. In B note the presence of abnormal matrix encircling disc cells and decreased matrix in intraterritorial regions. In Fig. [2C](#F2){ref-type="fig"} a region of prominent focal loss of matrix is present near encapsulated cells. (A, glycol methacrylate embedment, Goldner\'s stain; B and C, paraffin with Masson-trichrome; X 980).](1471-2474-3-9-2){#F2}

At the ultrastructural level, wavy collagen bands characterize regions of good matrix (Figure [3A](#F3){ref-type="fig"}). In more degenerated areas, collagen fiber number is much less (Figure [3B](#F3){ref-type="fig"}).

![Electron micrographs showing regions of collagen fibers in a wavy pattern in a region of annulus (Fig. [3A](#F3){ref-type="fig"}) which contrasts with sparser collagen in a region with more prominent matrix loss (Fig. [3B](#F3){ref-type="fig"}). (Surgical specimen from a 35 year old female. (Fig. [3A](#F3){ref-type="fig"}, X 5,900; Fig. [3B](#F3){ref-type="fig"}, X 13, 940).](1471-2474-3-9-3){#F3}

When viewed with polarizing light microscopy, regions of the disc with collagen crimping stand out prominently, and visualization of crimping patterns can be enhanced by application of specialized image analysis techniques which provide a graphical representation of image intensities (Figure [4](#F4){ref-type="fig"}). In regions of good disc morphology, collagen crimping patterns produce image intensity graphs where crimp patterns show clear peaks and valleys in register (Figure [4A](#F4){ref-type="fig"} and [4B](#F4){ref-type="fig"}). In areas where degenerative changes in the matrix are prominent, however, collagen crimping is markedly disrupted (Figure [4D](#F4){ref-type="fig"}). Note that the area examined in Figure [4D](#F4){ref-type="fig"} shows some crimp morphology (arrows), thus confirming that the plane of sectioning was appropriate for the detection of collagen crimping. Figure [4C](#F4){ref-type="fig"} shows that rare collagen bundles in register with each other are still present in this area, thus verifying that the tissue section plane was appropriate for detection of collagen crimping.

![Photomicrographs of disc tissue from the annulus in a region of healthy matrix (Fig. [4B](#F4){ref-type="fig"}, surgical specimen from a 46 year old female) and from a region with marked matrix changes (Fig. [4D](#F4){ref-type="fig"}, surgical specimen from a 54 year old male). In Figures [4A](#F4){ref-type="fig"} and [4C](#F4){ref-type="fig"}, collagen crimping patterns throughout the same microscopic fields shown in Figs. [4B](#F4){ref-type="fig"} and [4D](#F4){ref-type="fig"} are converted to graphic images with three-dimensional intensity images using BioScan^®^ OPTIMAS™ using the following settings for both images: rotation: x, 25.000; y, 1.000; sampling, x: 45, y:45, and viewpoint Z: 3000.0 . White arrows in Fig. [4D](#F4){ref-type="fig"} mark small regions showing collagen crimping, but degenerative changes in the tissue disrupt crimping in the adjacent regions. (Figs. [4B](#F4){ref-type="fig"} and [4D](#F4){ref-type="fig"}, polarized light, picrosirius red/alcian blue, X 102).](1471-2474-3-9-4){#F4}

Discussion
==========

In order for the disc to successfully withstand mechanical forces, its matrix must have the appropriate structural features, and this matrix must be maintained by appropriate turnover of its molecules. Previous studies from our laboratory have identified similar matrix changes during disc aging and degeneration in disc tissue from both control subjects and patients undergoing disc surgery \[[@B7],[@B8]\]. Observations presented here focus upon secondary collagen changes involving crimping morphology which nonetheless have important consequences for tissue biomechanical function.

During aging and degeneration of the intervertebral disc, disruption of annular fibers during stress can result in fissures which may extend out to the disc margin \[[@B9]\]. Such radial tears may be a source of the nonspecific early spinal symptoms in disc degeneration \[[@B9]\]. Although the importance of annular tears has long been recognized, the basis for these tears at the architectural level has not been investigated.

In the present work, study of a collection of disc specimens from control and surgical patients has shown that the architectural features of collagen crimping in the annulus can be disrupted in three fashions. First, a common morphologic change during disc aging/degeneration is the accumulation of wide layers of unusual matrix which encircle disc cells and clusters of disc cells \[[@B7],[@B8]\]. These regions disrupt normal collagen fibrillar layers and thus reduce the functional stretch afforded by collagen crimping in regions of normal collagen. Secondly, in areas where the matrix has become sparse, only a few collagen fibers are present. Such regions would have diminished capability of affording appropriate stretch function to resist tearing under compression. Finally, marked extracellular matrix loss in focal areas altered normal lamellar collagen architecture. The presence of similar types of changes in both control and surgical disc tissue is not surprising in light of previous studies by Boden et al \[[@B10],[@B11]\] in which degenerative changes were identified with MRI in lumbar and cervical discs of older asymptomatic subjects.

In other ultrastructural studies of disc tissue, we have reported considerable variation in cross-sectional diameters of collagen fibrils in both territorial and inter-territorial extracellular matrix \[[@B8]\]. Cross-sectional diameter variability was present in 34% of control and 59% of surgical specimens which were examined. Such variations may also have a possible effect on the ability of the disc to respond to biomechanical stress.

A limitation of the present study is the qualitative nature of our study. Future studies would be greatly strengthened by quantitative analysis of variations of crimping periodicity in both surgical specimens and in representative sections of complete discs. Such quantitative studies are important since histologic/ultrastructural examination only focuses upon a small portion of tissue. Additional important information lacking from our study is determination of the extent to which the changes described here actually alter the biomechanical properties of the disc. We hope that our small study here will lead to more complete studies of this important topic since there is much to be learned about how disc degeneration relates to increased risk of herniation and pain.

Little is understood about collagen turnover in either the healthy or diseased intervertebral disc. Studies by Antoniou et al have pointed out that it is very difficult to distinguish between synthesis of new matrix, degradation products, and resident matrix collagen \[[@B12]\]. It has been suggested that the turnover in the disc may be in excess of 100 years \[[@B9]\]. Newer methods used by Antoniou et al reveal that synthesis of Type I and II collagen and of aggrecan all decrease from age 2 to 15, but Type I collagen appears to decline more rapidly and to plateau during ages 15--40 years, whereas Type II collagen and aggrecan continue to decline during this period \[[@B12]\]. At older ages (60--80 years), there is increased denaturation of Type II collagen and a small increase in the synthesis of Type I procollagen. The maintenance of appropriate matrix composition in the disc is complicated by a marked decline in the number of disc cells with aging and an increase in cellular death by apoptosis \[[@B7]\].

Conclusions
===========

In summary, use of our large disc tissue collection was an advantage in the present study since it provided examination of numerous disc specimens sampled through various planes of sectioning. Application of image intensity graphs provided objective tracings of collagen patterns. Although proteoglycan loss has long been acknowledged to be an important part of disc aging and degeneration, the work presented here suggests that important stretch responses of collagen made possible by collagen crimping may be markedly altered by morphologic changes in disc architecture during aging/degeneration; such changes may contribute to the early tissue changes involved in annular tears. This work points to the need to more fully understand the dynamic relationship between degenerative changes in disc architecture at the microscopic level and resultant alterations in biomechanical function.

Authors\' contributions
=======================

ENH contributed disc tissue from surgeries on patients, and participated in the study and reviewed findings with HEG. HEG conceived of the study and participated in its design, coordination, reviewed all light and electron microscopic material, and wrote the manuscript. All authors read and approved the final manuscript

Competing interests
===================

None declared

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

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Acknowledgements
================

The authors thank Jane Ingram and Tracie McClain for assistance with light microscopy, and Daisy Ridings, Pat McCoy and Winston Wiggins for assistance with electron microscopy.