Retinal haemodynamics in individuals with well-controlled type 1 diabetes

Aims/hypothesis Abnormalities in retinal haemodynamics have been reported in patients with type 1 diabetes in advance of clinical retinopathy. These abnormalities could therefore be useful as early markers or surrogate endpoints for studying the microangiopathy. Since the DCCT, the increased focus on good glycaemic control is changing the natural history of diabetic retinopathy. Based on this, the aim of this study was to investigate whether patients with type 1 diabetes treated entirely or mostly in the post-DCCT era and tested in the absence of confounding factors show retinal haemodynamic abnormalities.
The DCCT proved that good glycaemic control is a powerful means of preventing retinopathy or slowing its course in many patients [1]. It also proved that today’s means of achieving good control are imperfect, and that some patients may still progress to sight-threatening stages of retinopathy. With the latter finding, the DCCT encouraged continuation of the quest for therapies able to pre-empt the effects of the residual hyperglycaemia; however, by increasing the prevalence of good glycaemic control it made any trial of such therapies extremely long and expensive. For this reason, there is heightened interest in early markers of retinopathy to be used as surrogates to test adjunct therapies.
Retinal haemodynamics have been investigated extensively in the quest for early functional markers of diabetic retinopathy [2]; some of the changes detected appear to be a function of the duration of diabetes and the severity of retinopathy [2–4]. In patients with type 1 diabetes and absent or minimal retinopathy, several investigators using different techniques have observed decreased retinal blood speed [3–7]. These studies, performed in the early and mid-1990s, were in patients with rather poor glycaemic control by today’s standards, yet the decreased retinal blood speed could not be attributed solely to elevated blood glucose levels at the time of measurement because hyperglycaemia actually increases blood speed [2, 3, 7]. We investigated whether patients with type 1 diabetes treated entirely or mostly in the post-DCCT era and tested in the absence of confounding factors show retinal haemodynamic abnormalities.
Methods
The institutional review boards of the Schepens Eye Research Institute and Massachusetts General Hospital (MGH) approved this study. We recruited, from the MGH Diabetes Center and the greater Boston area, type 1 diabetic patients with the clinical characteristics of patients previously reported [3–7] to show decreased retinal blood speed: 18–45 years of age, 1–15 years of insulin dependence, and absent or minimal retinopathy. The non-diabetic participants, matched for age and sex, had no history of diabetes and had HbA1c levels lower than 6%. Exclusion criteria were pregnancy, smoking, systemic diseases other than type 1 diabetes, other retinal diseases, hypertension and renal insufficiency; also excluded were users of angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, vitamin E at doses greater than 400 IU/day, aspirin or other antiplatelet drugs, and ibuprofen. After giving informed consent, all participants in the study underwent a complete eye examination. Diabetic individuals were excluded if they had diabetic retinopathy greater than level 20 in the Early Treatment Diabetic Retinopathy Study severity scale (microaneurysms only). HbA1c was measured by the MGH HbA1c laboratory; a level >10% was an exclusion criterion.
The retinal haemodynamic parameters were measured when capillary blood glucose levels were between 3.8 and 11.1 mmol/l in order to avoid the confounding effects of acute hypo- or hyperglycaemia on retinal blood flow [7]. The investigator performing the laser Doppler measurements (G. T. Feke) was masked to the diabetes status of the participants. The study eye was chosen on the basis of vascular anatomy. The superior temporal retinal arteries are preferred because temporal arteries are larger than nasal arteries, and the lesions of diabetic retinopathy are more prevalent in the superior temporal than in the other quadrants. The arterial blood column diameter and centreline blood speed were measured using a Canon laser Doppler retinal blood flow instrument (CLBF 100; Canon, Tokyo, Japan) [8]. The blood flow rate in units of μl/min is calculated automatically as the product of the cross-sectional area of the artery at the laser Doppler measurement site and the average blood speed, assuming a circular arterial cross-section [8].
Data are presented as means±SD. Parameters were compared between diabetic and control participants using the non-paired t test.
Results
Table 1 summarises the characteristics of the study population. The diabetic and control groups differed only in the levels of HbA1c and blood glucose. The diabetic participants received insulin through multiple daily injections or an external pump. The mean HbA1c level was 7.5% and the range 5.2–9.3%, eight patients having HbA1c levels above 8%. The mean duration of type 1 diabetes was 8.8 years. Only three patients manifested retinopathy, two of them showing one microaneurysm and one several microaneurysms. Of note, no diabetic individual who sought participation in the study was excluded because of advanced retinopathy, and only one diabetic individual was not included because of HbA1c greater than 10% (10.8%).
Table 1Characteristics of the study population Diabetic individuals (n = 33)Controls (n = 31)p valueAge (years)30 ± 7.030 ± 5.60.86Sex (% female)48580.44HbA1c (%)7.5 ± 1.25.0 ± 0.3<0.0001Diabetes duration (years)8.8 ± 4.6n/an/aBlood glucose level (mmol/l)a7.7 ± 2.85.4 ± 0.8<0.0001Systolic blood pressure (mmHg)106 ± 11109 ± 70.27Diastolic blood pressure (mmHg)65 ± 866 ± 70.47Retinopathyb (%)9n/aData are means±SD or percentagesaMeasured immediately before the retinal haemodynamic studiesbAssessed using the Early Treatment Diabetic Retinopathy Study severity scale. Individuals with a score of greater than 20 were excluded from the studyn/a, not applicable
Figure 1 presents the retinal haemodynamic measurements in 27 diabetic and 26 control participants in whom the measurements could be performed at the major superior temporal artery. Arterial diameter and blood speed were not significantly different between the diabetic patients and the controls (diameter: patients 116 ± 12 μm, controls 112 ± 10 μm, p = 0.18; blood speed: patients 32.9 ± 6.0 mm/s, controls 34.6 ± 6.8 mm/s, p = 0.34). Accordingly, retinal blood flow was the same in the two groups (patients 10.4 ± 2.4 μl/min; controls 10.4 ± 3.2 μl/min, p = 0.98). In the remaining six diabetic and five control participants there was early bifurcation of the artery, and one of the branches was used for the measurements; again, no differences were noted between the two groups. The sample size provided 80% power (α = 0.05) to detect differences as low as 0.75 SD between diabetic and control participants. This translates into differences of 7, 15 and 24% for retinal artery diameter, blood speed and blood flow, respectively.
Fig. 1Retinal haemodynamic parameters in type 1 diabetic individuals and matched non-diabetic controls. The box plots present the data for the 27 diabetic and 26 control participants in whom the measurements could be performed at the major superior temporal artery. Each box plot shows the 10th, 25th, 50th (median), 75th and 90th percentiles of the indicated parameter. Values above the 90th and below the 10th percentile are plotted as points
None of the haemodynamic measurements showed a significant correlation with coincident blood glucose levels, HbA1c, age, diabetes duration, systolic or diastolic blood pressure in either the diabetic or the control participants (data not shown).
Discussion
The important finding of this study is that in young patients with relatively well-controlled type 1 diabetes and no complications the baseline retinal circulatory parameters are within the normal range, even after several years of diabetes. These results differ from those of studies completed before the publication and widespread application of the DCCT results. In these earlier studies [3–7] the average HbA1c levels were 8, 10 or 12% compared with 7.5% in our diabetic individuals. Although we did not observe a correlation of HbA1c with retinal blood speed or flow, most likely because of the narrow range of HbA1c values, it was previously shown that retinal blood flow decreased with increasing HbA1c in type 1 diabetic patients without retinopathy [4].
The relation between HbA1c and retinal circulatory parameters must be interpreted with the knowledge that acute elevations [2, 7] or coincident high levels [2, 3] of blood glucose increase retinal blood flow, by increasing blood speed. Thus, the decreased blood speed and blood flow found in type 1 patients in previous studies indicated the presence of chronic effects of hyperglycaemia on retinal vessels in advance of clinical retinopathy. Elevated plasma glucose levels at the time of haemodynamic measurements could actually have masked, to some extent, such chronic effects of hyperglycaemia, contributing to the inconsistency with which retinal circulatory abnormalities were detected in diabetic patients without retinopathy [2]. The design of our study, requiring relatively normal glucose levels prior to the laser Doppler measurements (Table 1), limited such potential confounding effects, and the normal retinal haemodynamics that we recorded in diabetic participants should be informative of the status of retinal vessels.
Our results are empowering for patients with type 1 diabetes and those who care for them, for two reasons. First, it is apparent that good glycaemic control is attainable in a non-clinical trial setting, and is sustainable over time. Second, such good control not only delays or prevents retinopathy, as shown by the DCCT, but also delays or prevents abnormalities in the retinal circulation that may be prodromes to the development of retinopathy. Normal retinal haemodynamics were also reported recently in patients with type 2 diabetes with no retinopathy and mean HbA1c of 7.2% [9] or 7.7% [2].
From the standpoint of finding early markers of retinopathy, our observations indicate that measurements of retinal circulatory parameters in the unperturbed state may not be sufficiently sensitive in patients with absent or minimal retinopathy and well-controlled diabetes. Insofar as the HbA1c levels in such patients are often still greater than normal, the residual hyperglycaemia is likely to leave signs on the retinal vessels, but these may be uncovered only when a functional challenge is applied to the vessels. In response to hyperoxia, used as a stimulus to test vascular reactivity, patients with well-controlled type 2 diabetes showed normal retinal haemodynamics [9], but type 1 patients in another study did not [10]. Unfortunately, the latter study did not report levels of glycaemic control. The next step will be to compare in the same well-controlled type 1 diabetic individuals the behaviour of retinal haemodynamics at steady state and in response to a challenge. In this way we will begin to reconstruct systematically the history of retinal microangiopathy in the post-DCCT era.

type 1 diabetes
diabetic retinopathy
retinal blood flow
retinal circulation


Surgical management of dural arteriovenous fistulas with transosseous arterial feeders involving the jugular bulb

Dural arteriovenous fistulas located in the vicinity of the jugular foramen are complex vascular malformations and belong to the most challenging skull base lesions to treat. The authors comprehensively analyze multiple features in a series of dural arteriovenous fistulas with transosseous arterial feeders involving the jugular bulb. Four patients who underwent surgery via the transcondylar approach to treat dural arteriovenous fistulas around the jugular foramen were retrospectively reviewed. Previously, endovascular treatment was attempted in all patients. The success of the surgical treatment was examined with postoperative angiography. Complete obliteration of the dural arteriovenous fistulas (DAVFs) was achieved in three patients, and significant flow reduction in one individual. All patients had a good postoperative outcome, and only one experienced mild hypoglossal nerve palsy. Despite extensive bone drilling, an occipitocervical fusion was necessary in only one patient with bilateral lesions. The use of an individually tailored transcondylar approach to treat dural arteriovenous fistulas at the region of the jugular foramen is most effective. This approach allows for complete obliteration of the connecting arterial feeders, and removal of bony structures containing pathological vessels.
Introduction
Ten to 15% of intracranial arteriovenous fistulas are dural lesions [27], and these are most commonly related to dural venous sinuses [7]. Only a few previous articles have reported the less common intraosseous dural arteriovenous fistulas occurring at the anterior or posterior skull base [8, 19, 22, 26, 30]. Although the pathogenesis of dural arteriovenous fistulas (DAVFs) is not yet well understood, angiogenic factors and local hypoxia might play the most important roles in angiogenesis of DAVFs [24, 36–39]. The known experience suggests that the natural history of DAVFs is sometimes progressive, and intervention aiming at closure of these fisulas should be considered in selected cases—either surgically, endovascularly, or radiosurgically [6, 11, 15, 21]. In some circumstances, successful obliteration may not be possible by endovascular means, or patients may refuse the radiosurgical treatment. Direct surgery may be the best treatment option in such cases.
Surgical treatment of the DAVFs at the jugular foramen region aims at removal of the bony parts that contain the pathological vessels, and physical interruption or obliteration of the arterialised leptomeningeal venous connection. In the patients we have treated so far, the fistula complex was located within or in the vicinity of the occipital condyle, rendering the suboccipital transcondylar approach as the best access route for surgical treatment. Several previous studies related to fistulas with transosseous arterial feeders dealt with endovascular embolization or with a similar surgical technique, but in many aspects different from ours [14, 20, 21, 26]. It was the aim of the present study to detail our surgical technique for the treatment of DAVFs in the region of the jugular foramen, and to retrospectively review the clinical outcome of four patients treated at our institute during a 5-year period.
Patients and methods
Patients
Between June 1999 and February 2004, four patients underwent surgery at the Department of Neurosurgery, Philipps University for removal of DAVFs involving the jugular foramen. Patients ranged in age from 58 to 72 years, with a mean of 67 years. There were three females and one male; one individual had bilateral lesions. Four lesions were located on the right, and one on the left side. All patients were symptomatic, the most common presenting symptom being pulsatile bruit. The clinical details are summarized in Table 1.
Table 1Summary of clinical featuresCase no.Age/sexPresentationLocationEmbolizationAngiographyAttempts and intervalOutcome1. WP58Y/MPulsatile bruit, rt. hypoglossal nerve paresisRt. jugular process  and bulbYesFeeders: rt. ascending pharyngeal and occipital arteries of ECA2, 2 daysNo additional deficits; symptom free2. KW72Y/FPulsatile bruit, swelling of rt. upper and lower eyelidsRt. jugular process  and bulbYesFeeders: Both ascending pharyngeal and rt. occipital arteries of ECA4, 5 daysNo deficits; symptoms improved3. SH70Y/FPulsatile bruitDorsal rim of foramen magnum, both occipital condyles and jugular processesYesFeeders: Both internal maxillary and occipital arteries of ECA2, 1 monthNo deficits, symptom-free4. BE68Y/FPulsatile bruitRt. Jugular tubercle and bulbYesFeeders: rt. ascending pharyngeal and occipital arteries of ECA2, 4 daysMild hypoglossal nerve palsy; symptom-freeAbbreviations: Y: years; M: male; F: female; Rt: right; ECA: external carotid artery; Attempts and interval: attempts of endovascular embolization, and timing between the last endovascular and surgical treatment
All patients underwent preoperative angiography and at least two attempts of endovascular embolization. For the occlusion of arterial feeders, Guglielmi electrolytically detachable platinum coils were used. The decision for surgical intervention was made after the endovascular approach proved unsuccessful in terms of complete obliteration, and the patient developed neurological deficits or intolerable symptoms of pulsatile bruit.
Case histories
Case 1 A 58-year-old male presented with a 1-year history of pulsatile tinnitus in the right ear. Neurological examination revealed a partial right-sided hypoglossal palsy. Superselective angiography disclosed multiple DAVFs between the distal right sigmoid sinus and transosseous arterial feeders from the right ascending pharyngeal and right occipital arteries. Although the endovascular intervention resulted in coil occlusion of the occipital artery, the DAVFs still persisted (Fig. 1). Microsurgical intervention was performed due to intolerable pulsatile tinnitus, with the patient in the sitting position. Only minimal bony involvement around the jugular foramen was detectable on the CT scan (Fig. 1).
Fig. 1DAVFs with transosseous arterial feeders. Right external carotid angiogram, antero-posterior (AP) (a) and lateral (b) views, demonstrate arteriovenous fistulas (arrows) adjacent to the right internal jugular bulb. The fistulas are mainly fed by the right-sided ascending pharyngeal and occipital arteries. Symptoms were improved following transarterial embolization with coils (c), but the fistulas persisted. Intraoperative photograph with the patient in the sitting position showing the electrodes for lower cranial nerve monitoring (d). Postoperative angiograms (e and f) demonstrate cure of the DAVFs. The same patient’s axial CT scan reveals the preoperative intraosseous lesion near the jugular process (g). Postoperative CT scan shows minimal bony removal (h)
Case 2 This 72-year-old female had suffered from pulsatile tinnitus in the right ear for more than 18 years. During this period, the patient was angiographically diagnosed as having DAVFs in the region of the right jugular bulb. Four sessions of endovascular intervention were performed without success. Several months before admission, the patient experienced a clinical deterioration in the form of right periorbital swelling. In addition, there was excessive lacrimation of the right eye, and progressive numbness occurred around her right ear. On the CT scan, a bony erosion of the medial portion of the right occipital condyle and jugular tubercle became evident. A recent angiography disclosed the typical early filling of the right jugular bulb, and right sigmoid and transverse sinuses through intraosseous DAVFs mainly supplied by both ascending pharyngeal arteries and both occipital arteries (Fig. 2). The patient subsequently underwent surgical intervention in the left park bench position (Fig. 2).
Fig. 2Lateral (a) and AP (b) preoperative angiograms of the right common carotid artery (CCA), demonstrating the right intraosseous DAVFs (arrows). Postoperative right CCA angiogram revealing the remaining fistula (c). On the CT scan (d) bony erosion of the medial portion of the right jugular tubercle becomes evident (white arrows). Selective injection of preoperative right occipital artery reveals intraosseous fistula (e). The patient is positioned in the left lateral park bench position (f)
Case 3 A 70-year-old female presented with a 10-month history of bilateral pulsatile tinnitus predominant in the right ear. Neurological examination was irrelevant, and superselective angiography revealed numerous bilateral DAVFs in the foramen magnum region affecting both occipital condyles and jugular processes (Fig. 3). The fistula drained into both jugular veins, respectively. Feeding arteries were branches from the internal maxillary and occipital arteries. After partial occlusion of feeders on the right side, the intensity of the tinnitus decreased but still persisted. The patient was severely affected by this symptom. Surgery was therefore carried out using a right transcondylar exposure, and post-operatively the patient reported further improvement, with minimal residual tinnitus. During a 6-month follow-up, the intensity of left pulsatile bruit increased. Additionally, the patient developed a movement-related neck pain which proved to be caused by a fracture of the right occipital condyle, with dislocation of a bony fragment and craniocervical instability (Fig. 3). She underwent a second surgical procedure via a left transcondylar approach to treat the left DAVFs, followed by an occipitocervical fusion performed in the same surgical setting (Fig. 4).
Fig. 3Bilateral intraosseous DAVFs causing bilateral pulsatile tinnitus. Right lateral (a) and AP (b) external carotid angiograms, as well as right AP (c) common carotid angiogram demonstrate multiple arteriovenous shunts fed by the right internal maxillary and occipital arteries. MRI (d) and CT (e) studies show the abnormal vessels posterior to the right jugular tubercle and the enlarged jugular foramen with its surrounding bony erosion, respectively (arrows). The photograph shows the skin incision for the right transcondylar approach (f). Intraoperative screenshot of navigation, applied for localizing the exent of the arteriovenous fistulas (g)Fig. 4Postoperative CT scan of the same patient shown in Fig. 3 reveals a fracture of the right occipital condyle with dislocation of a bony fragment (a). Postoperative angiograms of the right external carotid artery (ECA), lateral (b) and AP (c) views showing no arteriovenous fistula after the surgical resection of right intraosseous DAVFs. Preoperative angiogram of the left ECA revealing arterial filling of the jugular vein and left-side DAVFs (d). The patient is placed in the prone position for the surgery of the left intraosseous DAVFs and the occipito-cervical fusion (e). Postoperative lateral plain radiogram (f) and a schematic drawing (g) demonstrating the instrumentation used for the occipitocervical fusion (g)
Case 4 This 68-year-old female complained of progressive pulsatile right-sided tinnitus for 1 year. Neurological examination was irrelevant. An angiogram obtained at our institution furnished evidence of DAVFs at the right occipital condyle and jugular tubercle. The fistula was mainly fed by meningeal branches of the right occipital and ascending pharyngeal arteries. The patient underwent embolization of the right occipital artery, aimed at reducing the arterial supply. Due to persistent arteriovenous connection and impairment of the patient’s quality of life, surgery was subsequently performed.
Surgical technique
All surgical procedures were performed by the senior author (H.B.). The first aim of surgery was an adequate exposure of the dorsolateral suboccipital region, including the medial portion of the occipital condyle, the lateral rim of the foramen magnum, the arch of the atlas and the horizontal portion of the vertebral artery, as well as the medial mastoid and particularly the jugular process region (Fig. 5). The patient was placed either in the sitting or in the lateral park bench position with the head flexed, turned to the ipsilateral and slightly tilted to the contralateral side. It was considered important to reduce the venous congestion by avoiding jugular-vein compression or by elevating the head when the lateral park bench position was chosen. Arterial feeders were occasionally encountered during muscular dissection in the course of the occipital artery in those patients in whom the artery has not been completely obliterated endovascularly before surgery.
Fig. 5The particular arteriovenous fistulas are located within the occipital condyle and the jugular foramen region. These fistulas are fed by dural branches of both internal and external carotid arteries (upper). The drawing demonstrates the intraosseous fistulas, arterialized diploic veins and draining vein (lower)
First, a small dorso-lateral suboccipital craniectomy was performed, which basically corresponds to a dorso-lateral enlargement of the foramen magnum. Bone drilling began in the supracondylar fossa, and was extended both medially towards the rim of the foramen magnum and laterally towards the mastoid. By gradually exposing the distal sigmoid sinus and the dura mater of the suboccipital and upper cervical region, transosseous arterial feeders were encountered, which occasionally caused a brisk arterial bleeding (Fig. 6). To control this bony bleeding, drilling was temporarily performed using the diamond burrs without saline irrigation (Aesculap drills). Application of bone wax or bipolar coagulation to stop the bleeding was usually ineffective. In the patients with severe bony involvement, the consistency of the cancellous bone was remarkably altered. In these cases the bone marrow was packed with Surgicel and then slightly compressed with cottonoid to control the bleeding. In some cases, the arteriovenous fistulas involved the posterior condylar emissary vein located within/around the posterior condylar canal (Fig. 7). These bony portions were completely drilled away. While continuing drilling within the occipital condyle, the hypoglossal canal and the medial portion of the jugular bulb were gradually exposed. The medial wall of the jugular bulb, being the site of the arteriovenous fistulas, was coagulated to interrupt small arterial feeders of this area (Fig. 6).
Fig. 6Artistic illustration showing the transcondylar approach as applied to interrupt the connecting arterial feeders; the extent of the bony removal is judged according to the extent of the arteriovenous fistulas. Transosseous arterial feeders are encountered, which may occasionally cause a brisk arterial bleedingFig. 7Schematic drawing of intraosseous dural arteriovenous fistulas in the right hypoglossal canal and in the vicinity of the jugular foramen region. The feeders are the dural branches of the ascending pharyngeal artery. These arterial feeders connect with the inferior petrosal sinus, the jugular bulb and the posterior condylar emissary vein. (1: right jugular bulb, 2: right posterior emissary vein, 3: right inferior petrosal sinus, 4: arterialized venous plexus within the right hypoglossal canal, 5: right ascending pharyngeal artery, a: right internal auditory canal, b: pars nervosa of the right jugular foramen, c: right hypoglossal canal, d: right occipital condyle, e: anterior rim of the foramen magnum)
Intraosseous DAVFs were also encountered around the hypoglossal canal, with arteriovenous shunts flowing into the venous plexus surrounding the hypoglossal canal, and more anteriorly in the region of the inferior petrosal sinus. To reach this area, the bony resection was extended into a deeper region. Use of the intraoperative microdoppler probe gave a good impression of the arterialization of the jugular bulb. Thus, persistence of intraosseous arterial feeders indicated that bony resection had to be continued by drilling the jugular tubercle, the occipital condyle and the jugular process. The required extent of bone removal was also estimated from preoperative imaging, and was related to anatomical landmarks such as the hypoglossal canal, the posterior condylar emissary canal and the dural entrance of the vertebral artery. Drilling was stopped only when no more arterial feeders were encountered, indicating that all feeders had been interrupted. Normalization of blood flow within the jugular bulb could be documented by using the microdoppler probe. To adequately drill the dorsomedial portion of the occipital condyle, a small portion of the lateral atlantal mass had to be resected in some instances as well. In such cases, complete exposure of the horizontal portion of the vertebral artery was necessary. However, there was no need for complete C1 hemilaminectomy. The suboccipital dura mater was not opened, since an intradural inspection of the jugular foramen was unnecessary as there were no intradural arteriovenous shunts. After meticulous extradural hemostasis, wound closure was obtained in several layers.
Results
All four patients underwent surgical obliteration of their DAVFs via the transcondylar approach. This access route provided a good exposure of the target site, and allowed occlusion of the intraosseous pathological vessels. There were no intra-operative complications, and no permanent major postoperative deficit occurred. Despite a certain blood loss during surgery, no patient required intra-operative or postoperative blood transfusion. Although an extensive resection of the involved occipital condyle, jugular process and jugular tubercle was carried out in all patients, an occipito-cervical fusion was necessary only in patient 3 with bilateral lesions. There were no additional neurological deficits, with the exception of patient 4, who experienced mild hypoglossal nerve palsy postoperatively.
Postoperative angiography was obtained in each patient to confirm obliteration of the DAVFs. Three patients showed radiological cures, with no further treatment necessary. In patient 2, the DAVFs were significantly reduced surgically, but still persisted. Therefore, a second surgical intervention was proposed to the patient, who eventually refused. While preoperative complaints disappeared in three patients, they were clearly improved in one individual.
Discussion
Definition, terminology, pathology and symptoms
Dural arteriovenous fistulas [DAVFs] are defined as abnormal arteriovenous connections occurring within the leaflets of the dura mater, usually within or near the walls of a dural sinus [13]. Although the term “dural arteriovenous malformations” [DAVMs] has been used widely in the neuroscience literature, current nomenclatures include terms such as “DAVFs” and “dural arteriovenous shunts”, according to an accepted view that many of these lesions are acquired [7, 9, 10, 18]. Initially, DAVFs were described only in association with venous sinuses. Subsequently, they have been found in other locations such as the tentorium, the skull base and the intraorbital area as well [30, 31]. Piske and Lasjaunias hypothesized that DAVFs can develop wherever veins have their course [31]. This would explain why DAVFs can be located entirely within the bone, because emissary veins have transosseous courses. The particular group of dural arteriovenous fistulas located within the occipital condyle and jugular foramen region are intracavarial (intracranial) arteriovenous shunts fed by dural branches of both internal and external carotid arteries. Some authors have named this particular lesion “intraosseous DAVMs” [26] or “intraossous DAVFs” [22]. The fistulas are located within the bony structures surrounding the jugular bulb (the major draining vein), and may involve the adjacent draining veins [8, 26].
DAVFs involving the transverse and sigmoid sinuses always cause a pulsatile tinnitus [28, 29]. It seems that this symptom is related to venous turbulence in contact with the petrous bone, or to the high shunt flow into the petrosal sinus [22]. This might explain why all our patients presented with bruit as the predominant symptom.
Imaging
DAVFs are often invisible on CT and MR studies [17, 23]. Consequently, a normal contrast-enhanced CT or MR study does not exclude a DAVF. Contrary to the ordinary DAVFs, CT and MRI studies aid in giving the diagnosis of this particular DAVFs when the patient presents with pulsatile tinnitus due to demonstrable abnormal vessels in the soft tissue beneath the skull base [12, 32], or when bony erosion due to intraosseous abnormal vessels is present, as in the patients of our series. However, cerebral angiography remains the most important modality in evaluating DAVFs.
General management and endovascular intervention
According to current opinion, no single treatment is ideal for the obliteration of DAVFs, and management requires an interdisciplinary team approach which must be individualized for each patient. Malik et al. reported two patients with intraosseous dural arteriovenous fistulas located at the foramen magnum. He postulated that the intraosseous nature was related to the large number of emissary veins in this region, and recommended the exploration of the adjacent bone until the entire fistula is exposed [26]. Our observations and intraoperative findings confirm this operative experience, that considerable but controllable hemorrhage can occur during bone removal.
As being less invasive than surgery, endovascular technique is given preference as the primary treatment. However, transvenous embolization or transarterial coil embolization of the ascending pharyngeal artery during treatment of hypoglossal DAVFs may sometimes be associated with complications [14, 22]. An alternative surgical therapy is warranted if the endovascular technique eventually fails, as it happened to all patients of this study. The reason for failure of the endovascular intervention is probably the great number of arterial feeders that traverse through the occipital condyle and jugular process to enter the jugular bulb. A transvenous embolization was not carried out in our patients, to avoid the risk of major venous occlusion. Although stereotactic radiosurgery either followed or not followed by transarterial particulate embolization of accessible external carotid feeding vessels became the primary mode of treatment at some institutions [6, 15, 25, 33], it may not always replace open surgery. Radiosurgery is expected to cause obliteration of DAVFs between 1 and 3 years after treatment [6], and it is not considered as a standard therapy for all types of DAVFs. Benign cranial DAVFs [6, 34], though, are a developing indication for radiosurgery [34]; stereotactic radiosurgery may be considered as an alternative treatment for this type of DAVFs that have failed other treatments [20].
Surgical technique and avoidance of complications
In the majority of our previously treated patients, we have used the juxtacondylar/transcondylar approach to expose various intradural craniospinal lesions [1–5, 16]. To treat DAVFs, this approach was slightly modified, and primarily applied with the purpose of interrupting the pathological arterial feeders. The extent of bony resection at the skull base was, therefore, individually tailored according to the extent of arteriovenous fistulas under intraoperative microdoppler guidance.
The use of this surgical procedure was most effective, as all patients in our study showed postoperative clinical improvement, and complete obliteration of the intraosseous DAVF in three patients. Only one patient (patient 3), whose angiogram revealed residual AV fistulas, experienced previously developed mild periorbital edema. We believe that this was due to residual pathological vessels anterior to the jugular bulb, an area very difficult to reach surgically. During the removal of pathological bone containing abnormal fistulas in this patient, the dilated thin wall of the jugular bulb was slightly injured. After packing this area of venous bleeding with muscle fascia, no space for further drilling was available. A second, slightly-modified surgical intervention was, therefore, proposed to the patient to obliterate the remaining fistulas, but she refused.
Partial resection of the occipital condyle in DAVFs may be associated with the risk of injury to the vertebral artery and/or jugular bulb, or damage to the hypoglossal nerve. Since the arteriovenous fistulas may be located within and around the hypoglossal canal as in patient 4 of our series, the rootlets of the nerve may be injured by surgical manipulation, even in the hands of a very experienced neurosurgeon. Preoperative imaging with thin-slice CT scans or image fusion technique (which was applied in the last three patients) [35] can provide valuable information about the local topographical anatomy. This information comprises the variable anatomical configuration of the jugular tubercle, the course of the canal containing the posterior condylar emissary vein, and the size and exact location of arteriovenous fistulas.
Usually, the partial resection of the occipital condyle with opening of the atlanto-occipital joint does not create atlanto-occipital instability [2–5]. Only in the case of extensive bony drilling within the condyle, necessary due to the large number of transosseous pathological feeders, a partial condylar fracture may occur as it happened in patient 3 of this series. However, an occipito-cervical fusion was undertaken in this patient only because she harbored bilateral intraosseous DAVFs, and a second procedure on the opposite side was required that would have significantly destabilized the atlanto-occipital region.
Conclusion
Dural arteriovenous fistulas located around the jugular foramen are a less common type of DAVFs. The suboccipital transcondylar approach is considered the best access route when surgery of this particular pathology is intended. The amount of pathological bone that must be removed by drilling depends upon the extent of transosseous arterial feeders. Surgical obliteration of this complex vascular malformation is possible, at least in the cases in which the arterial feeders do not extend too far anterior to the jugular bulb. With this surgical technique, an atlanto-occipital instability may occur in very large or bilateral lesions, requiring occipito-cervical fusion.

dural arteriovenous fistula
transosseous arterial feeder
jugular bulb
transcondylar approach
occipital condyle
intraosseous davfs


Downregulated parafibromin expression is a promising marker for pathogenesis, invasion, metastasis and prognosis of gastric carcinomas

Parafibromin is a protein encoded by the hyperparathyroidism 2 oncosuppressor gene and its downregulated expression is involved in pathogenesis of parathyroid carcinomas. To clarify the roles of parafibromin expression in tumourigenesis and progression of gastric carcinomas, it was examined by immunohistochemistry (IHC) on tissue microarray containing gastric carcinomas (n = 508), adenomas (n = 45) and gastritis (n = 49) with a comparison of its expression with clinicopathological parametres of carcinomas. Gastric carcinoma cell lines (MKN28, AGS, MKN45, KATO-III and HGC-27) were studied for parafibromin expression by IHC and western blot. Parafibromin expression was localised in the nucleus of gastric epithelial cells, adenoma, carcinoma cells and cell lines. Its expression was gradually decreased from gastritis to gastric carcinoma, through gastric adenomas (p < 0.05) and inversely correlated with tumour size, depth of invasion, lymphatic invasion, lymph node metastasis and Union Internationale Contre le Cancer (UICC) staging (p < 0.05) but not with sex or venous invasion (p > 0.05). Parafibromin was strongly expressed in older carcinoma patients compared with younger ones (p < 0.05). There was stronger positivity of parafibromin in intestinal-type than diffuse-type carcinomas (p < 0.05). Univariate analysis indicated cumulative survival rate of patients with positive parafibromin expression to be higher than without its expression (p < 0.05). Multivariate analysis showed that age, tumour size, depth of invasion, lymphatic invasion, lymph node metastasis, UICC staging and Lauren’s classification but not sex, venous invasion or parafibromin expression were independent prognostic factors for carcinomas(p < 0.05). Downregulated parafibromin expression possibly contributed to pathogenesis, growth, invasion and metastasis of gastric carcinomas. It was considered as a promising marker to indicate the aggressive behaviours and prognosis of gastric carcinomas.
Introduction
Parafibromin is a protein encoded by the hyperparathyroidism 2 (HRPT2) oncosuppressor gene, whose mutation causes the hyperparathyroidism–jaw tumour syndrome. The disease is an autosomal dominant disorder characterised by the occurrence of parathyroid adenoma or carcinoma, fibro-osseous jaw tumours of the mandible or maxilla and renal neoplastic and non-neoplastic abnormalities, such as Wilms’ tumour, hamartoma or cystic renal disease [1, 16, 20]. HRPT2 gene is located in human chromosome 1q31.2, consists of 17 exons and spans 18.5 kb in the genome. It encodes a 2.7-kb transcript which is translated into a 531-amino-acid parafibromin protein with a molecular weight of 60 kd [3, 4, 23]. The 200-amino-acid C-terminal segment of parafibromin shares 32% identity and 54% homology with cell division cycle 73, a Saccharomyces cerevisiae protein forming the polymerase-associated factor 1 (Paf1) complex, which is associated with ribonucleic acid (RNA) polymerase II and involved in transcript site selection, transcriptional elongation, histone H2B ubiquitination, histone H3 methylation, poly (A) length control and coupling of transcriptional and posttranscriptional events [10, 17, 18, 24, 27]. Parafibromin overexpression was documented to inhibit colony formation and cellular proliferation and induce cell cycle arrest in the G1 phase, indicating that parafibromin has a critical role in cell growth [28]. Northern blot analysis showed HRPT2 expression in heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas [4]. Western blot study revealed parafibromin expression as a 60-kd band in the adrenal gland, heart, pancreas and kidney but 40-kd immunoreactive bands in the heart and skeletal muscle of human [25]. Immunohistochemically, higher expression of parafibromin was found widespread in glomerular mesangial cell, hepatocytes, cells of the base of gastric glands, renal cortex tubules and the pars intermedia of the hypophysis [17]. Subsequent investigations have revealed that mutations in HRPT2 are present in 66–100% of sporadic parathyroid carcinomas [7, 20]. Hyperparathyroidism–jaw-tumours-syndrome-related and sporadic parathyroid carcinomas are characterised by loss of parafibromin nuclear immunoreactivity [5, 22]. Selvarajan et al. [19] found that parafibromin expression was inversely linked to tumour size, pathologic stage and lymphovascular invasion of breast carcinomas using immunohistochemistry in a tissue microarray (TMA) study. These findings suggested the potential roles of parafibromin in pathogenesis and progression of malignancies.
Gastric carcinoma ranks as the world's second leading cause of cancer mortality behind lung cancer despite a sharp worldwide decline in both its incidence and mortality since the second half of the 20th century [9]. Tumourigenesis and progression of gastric carcinoma is a multistage process with the involvement of a multifactorial aetiology, which mainly results from gene–environment interactions [32, 33]. Gastric carcinomas are classified into early and advanced ones on the basis of whether the carcinomas invade into the muscularis propria of the stomach [12]. In 1965, Lauren [13] classified gastric carcinomas into intestinal- and diffuse-type ones based on the morphological appearances. Intestinal-type carcinomas are characterised by cohesive carcinoma cells forming gland-like tubular structures with expanding or infiltrative growth pattern. However, the cell cohesion is less apparent or absent in diffuse-type carcinoma and cancer cells diffusely spread in the gastric wall [31]. Generally, there is a favorable prognosis for the patients with early or intestinal carcinoma compared with the other type. In our study, parafibromin expression was examined in gastric carcinoma, adenoma, gastritis and gastric carcinoma cell lines and compared with the clinicopathological parametres of carcinomas, as well as prognosis to explore the clinicopathological significance and molecular roles of parafibromin expression in stepwise development of gastric carcinoma.
Materials and methods
Subjects
Gastric carcinomas (n = 508) were collected from the surgical resection, adenoma (n = 45) from endoscopic biopsy or polypectomy and gastritis (n = 49) from the endoscopic biopsy in our affiliated hospital, Himi Citizen Hospital and Kouseiren Takanoka Hospital between 1993 and 2006. All carcinomas were adenocarcinomas and the adenoma group was free from non-neoplastic polyp types, leiomyomas and benign gastrointestinal stromal tumours. The patients with gastric carcinoma were 354 men and 154 women (29~91 years, mean = 65.4 years). Among them, 191 cases have carcinomas accompanied with lymph node metastasis. None of the patients underwent chemotherapy or radiotherapy before surgery. They all provided consent for use of tumour tissue for clinical research and our University Ethical Committee approved the research protocol. We followed up all patients by consulting their case documents or through telephone.
Pathology
All tissues were fixed in 4% neutralised formaldehyde, embedded in paraffin and incised into 4-μm sections. These sections were stained by haematoxylin and eosin (HE) to confirm their histological diagnosis and other microscopic characteristics. The staging for each gastric carcinoma was evaluated according to the Union Internationale Contre le Cancer (UICC) system for the extent of tumour spread [21]. Histological architecture of gastric carcinoma was expressed in terms of Lauren’s [13, 31] classification. Furthermore, tumour size, depth of invasion, lymphatic and venous invasion were determined.
Tissue microarray
Representative areas of solid tumours were identified in HE-stained sections of the selected tumour cases and a 2-mm-in-diametre tissue core per donor block was punched out and transferred to a recipient block with a maximum of 48 cores using a Tissue Microarrayer (AZUMAYA KIN-1, Japan). Four-micrometre-thick sections were consecutively incised from the recipient block and transferred to polylysine-coated glass slides. HE staining was performed on TMA for confirmation of tumour tissue.
Cell lines and culture
Gastric carcinoma cell lines come from the Japanese Physical and Chemical Institute, including MKN28 (well-differentiated adenocarcinoma), AGS (moderately differentiated adenocarcinoma), MNK45 (poorly differentiated adenocarcinoma), KATO-III (poorly differentiated adenocarcinoma) and HGC-27 (undifferentiated adenocarcinoma). They were maintained in Roswell Park Memorial Institute 1640 (MKN28, MKN45 and KATO-III), minimum essential (HGC-27) or Ham’s F12 (AGS) medium supplemented with 10% foetal bovine serum, 100-units/ml penicillin, and 100-μg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C. Total protein was prepared from all cells by cell disruption buffer according to Protein And RNA Isolation System manual (Arctiris Bioscience, USA). All cells were collected by centrifugation, rinsed with phosphate-buffered saline, fixed by 10% formalin and then embedded in paraffin as routinely processed.
Immunohistochemistry
Consecutive sections were deparaffinised with xylene, dehydrated with alcohol and subjected to antigen retrieval by irradiating in target retrieval solution citrate pH 6.0 (TRS, DAKO, Carpinteria, CA 93013, USA) for 15 min with microwave oven (Oriental Rotor Lmt. Co., Tokyo, Japan). Five percent bovine serum albumin was then applied for 1 min to prevent non-specific binding. The sections were incubated with mouse anti-parafibromin antibody (Clone 2H1, SC-33638, Santa Cruz, CA, USA; 1:40) for 15 min, then treated with the anti-mouse Envison-PO (DAKO, CA, USA) antibody for 15 min. Binding sites were visualised with 3, 3′-diaminobenzidine with the 5-min reaction. All the incubations were performed in a microwave oven to allow intermittent irradiation as described previously [11]. After each treatment, the slides were washed with Tris-buffered saline with Tween 20 (TBST; 10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20) three times for 1 min. After being counterstained with Mayer’s haematoxylin, the sections were dehydrated, cleared and mounted. Omission of the primary antibody was used as a negative control.
One hundred cells were randomly selected and counted from five representative fields of each section blindly by three independent observers (Takano Y, Li XH and Zheng HC). The positive percentage of counted cells was graded semi-quantitatively according to a four-tier scoring system: negative (−), 0~5%; weakly positive (+), 6~25%; moderately positive (++), 26~50%; and strongly positive (+++), 51~100%.
Western blot
Fifty-microgramme denatured protein was separated on an SDS-polyacrylamide gel (10% acrylamide) and transferred to Hybond membrane (Amersham, Germany), which was then blocked overnight in 5% milk in TBST. For immunoblotting, the membrane was incubated for 1 h with mouse anti-parafibromin antibody as described above. Then, it was rinsed by TBST and incubated with anti-mouse immunoglobulin G conjugated to horseradish peroxidase (DAKO, CA, USA, 1:1,000) for 1 h. Bands were visualised with X-ray film (Fujifilm, Japan) by ECL-Plus detection reagents (Amersham, Germany). After that, membrane was washed with WB Stripping Solution (pH 2–3, Nacalai, Tokyo, Japan) for 30 min and treated as described above except mouse anti-β-actin antibody (Sigma, MO, USA, 1:5,000) as an internal control.
Statistical analysis
Statistical evaluation was performed using Spearman correlation test to analyse the rank data. Kaplan–Meier survival plots were generated and comparisons between survival curves were made with the log–rank statistic. The Cox’s proportional hazards model was employed for multivariate analysis. P < 0.05 was considered as statistically significant. SPSS 10.0 software was employed to analyse all data.
Results
Parafibromin expression in gastric tumours and carcinoma cell lines
As shown in Fig. 1, parafibromin was positively immunostained in the nucleus of MKN28, AGS, MKN45, KATO-III and HGC-27, and its expression level was consistent with the data of Western blot. Parafibromin was strongly expressed in the nucleus of gastric epithelial cells, adenomas and early carcinomas but not in given advanced carcinomas. Occasionally, it also appeared in stromal fibroblasts and lymphocytes but much weaker than epithelial cells or adenomas (Fig. 2).Generally, the stromal lymphocytes and fibroblasts were negative in cases where the tumour was negative. Overall, parafibromin expression was detected respectively in all gastritis (100.0%), 36 out of 45 adenoma patients (80.0%) and 233 out of total 508 gastric carcinoma patients (45.9%). Statistically, gradually reduced expression of parafibromin was seen from gastritis to gastric carcinoma through gastric adenoma (p < 0.05, Table 1). As summarised in Table 2, parafibromin expression was inversely correlated with tumour size, depth of invasion, lymphatic invasion, lymph node metastasis and UICC staging (p < 0.05) but not with sex or venous invasion (p > 0.05). Parafibromin was strongly expressed in older carcinoma patients compared with younger ones (p < 0.05). Intestinal-type carcinomas exhibited more frequent expression of parafibromin than diffuse-type ones (p < 0.05).
Fig. 1Parafibromin expression in gastric carcinoma cell lines. a Parafibromin was positively immunostained in the nucleus of MKN28 (a), AGS (b), MKN45 (c), KATO-III (d) and HGC-27(e). b Cell lysate (50 µg) was loaded and probed with anti-human parafibromin antibody (60 kd) with β-actin (42 kd) as an internal control. Lane #1: MKN28; #2 AGS; #3 MKN45; #4 KATO-III; #5 HGC-27Fig. 2Immunohistochemical staining of parafibromin in gastritis, adenoma and carcinoma. Note parafibromin positivity was strongly observed in the nucleus of gastric superficial epithelium (a), and adenoma (c) and early gastric carcinoma (b), occasionally weaker in the stromal fibroblasts and lymphocytes(a, c), but not in given advanced gastric carcinomas (d), indicating that the internal positive control (stromal cells) was negative adjacent to the negative staining carcinoma cells but positive adjacent to the positive epithelial cellsTable 1Parafibromin expression in gastric tissue samplesGroupsNumberParafibromin expression−++++++PR (%)Gastritis4901741100.0aGastric adenoma459582380.0bGastric carcinoma508275556011845.9PR Positive rateaCompared with gastric adenoma or carcinoma, p < 0.001bCompared with gastric carcinoma, p < 0.001Table 2 Relationship between parafibromin expression and clinicopathological features of gastric carcinomasClinicopathological featuresNumberParafibromin expression−++++++PR (%)Rsp valueAge (years)  <6520912524243640.20.095<0.05  ≥6529915031368249.8Sex  Male35418837389146.90.054>0.05  Female1548718222743.5Tumour size (cm)  <426311626348555.9−0.237<0.001  ≥424515927263335.1Depth of invasion  Tis−126310230409161.2−0.344<0.001  T2-424517325202729.4Lymphatic invasion  −33115742458752.6−0.168<0.001  +17711813153133.3Venous invasion  −443236495110746.7−0.051>0.05  +6539691140.0Lymph node metastasis  −31713838449756.5−0.285<0.001  +19113717162128.3UICC staging  0–I29212336419257.9−0.292<0.001  II-IV21615219192629.6Lauren’s classification  Intestinal-type27310834389360.4−0.322<0.001  Diffuse-type22515721222530.2PR Positive rate, Tis carcinoma in situ, T1 lamina propria and submucosa, T2 muscularis propria and subserosa, T3 exposure to serosa, T4 invasion into serosa, UICC Union Internationale Contre le Cancer
Univariate and multivariate survival analysis
Follow-up information was available on 508 gastric carcinoma patients for periods ranging from 0.2 months to 12.2 years (median = 67.2 months). Figure 3 showed survival curves stratified according to parafibromin expression for gastric carcinomas. Univariate analysis using the Kaplan–Meier method indicated cumulative survival rate of patients with weak, moderate or strong parafibromin expression to be obviously higher than without its expression (p < 0.05; Fig. 3a). The significant difference disappeared if stratified according to the depth of invasion (Fig. 3b, c). Multivariate analysis using Cox’ s proportional hazard model indicated that age, tumour size, depth of invasion, lymphatic invasion, lymph node metastasis, UICC staging and Lauren’s classification (p < 0.05) but not sex, venous invasion or parafibromin expression were independent prognostic factors for overall gastric carcinomas (p > 0.05; Table 3).
Fig. 3Correlation between parafibromin status and prognosis of the gastric carcinoma patients. Kaplan–Meier curves for cumulative survival rate of patients with gastric carcinomas according to the parafibromin expression in overall (a), early (b, EGC) and advanced (c, AGC) gastric carcinomasTable 3Multivariate analysis of clinicopathological variables for survival with gastric carcinomasNumberClinicopathological parametresRelative risk (95%CI)p valueAAge (≥65 years)1.929 (1.357–2.743)<0.001BSex (female)1.463 (0.983–2.179)>0.05CTumour size (≥4 cm)1.606 (1.013–2.547)<0.05DDepth of invasion (T2–4)6.530 (3.110–13.710)<0.001ELymphatic invasion (+)2.626 (1.516–3.374)<0.001FVenous invasion (+)0.959 (0.633–1.452)>0.05GLymph node metastasis (+)2.773 (1.525–5.043)<0.01HUICC staging (II-IV)0.294 (0.139–0.622)<0.01ILauren’s classification (diffuse-type)1.796 (1.212–2.661)<0.01JParafibromin expression (+~+++)0.792 (0.529–1.187)>0.05CI Confidence interval, UICC= Union Internationale Contre le Cancer
Discussion
HRPT2 has been isolated from complementary DNA libraries of parathyroid, kidney and bone tissue and encodes tumour suppressor protein parafibromin [4]. In the present study, the nuclear expression pattern was observed in the gastric epithelial cells, adenomas, adenocarcinomas and carcinoma cell lines consistent with previous reports in the gastric superficial mucosa, hepatocytes, kidney cortex tubules, adrenal gland, spleen lymphocytes, parathyroid tissue, adenoma and carcinomas, breast carcinoma [5, 8, 17, 19]. Although the result was in contrast with the paper of Porzionato et al. [17] possibly due to different incubation times of primary antibody, the great majority of immunohistochemical and cell transfection studies supported our observation of the nuclear staining [2, 5, 6, 8, 14, 19]. This study again demonstrates that parafibromin is nuclear and not cytoplasmic or nucleocytoplasmic in location as initially thought. The weaker expression of parafibromin in stromal cells than epithelial cells and adenoma might be due to the specificity of its cellular distribution as described previously [17]. It was found that the translocation of parafibromin to the nuclear compartment involved a function monopartite nuclear localisation signal at residues 136–139 [2, 6]. Parafibromin is a component of Paf1 complex in the nucleus, where it plays a role in cell cycle regulation, histone methylation, lipid and nucleic acid metabolism [10, 17]. The distribution pattern of parafibromin protein in gastric epithelial cells or tumour cells demonstrated its biological function in the nucleus.
Statistically, parafibromin expression was gradually reduced from gastritis to carcinoma through adenoma in line with parathyroid carcinogenesis, suggesting that downregulated parafibromin expression might contribute to the malignant transformation of gastric epithelial cells as an early event. The positive rate of parafibromin expression was reduced to 80% in gastric adenoma and reached about 46% of gastric adenocarcinoma, supporting the involvement of parafibromin in the gastric adenoma–adenocarcinoma sequence. Actually, the adenoma can progress into and be incorporated with gastric well-differentiated carcinoma when it grows bigger and de novo carcinogenesis is well understood, especially in diffuse-type gastric carcinomas [34]. Higher parafibromin expression in adenoma and intestinal-type carcinoma indicated that decreased parafibromin expression might play an important role in de novo diffuse-type carcinogenesis but less in intestinal carcinogenic pathway.
A body of evidences indicated that downregulation of tumour suppressor protein expression was due to genetic or epigenetic changes, like allelic loss, mutation, loss of heterozygosity (LOH), hypermethylation and microsatellite instability in malignancies [29, 30]. In the sporadic parathyroid carcinomas and hyperparathyroidism–jaw tumours, LOH or mutation of HRPT2 might cause the loss and inactivation of parafibromin protein [20, 25, 26]. Furthermore, the reduced expression of parafibromin was found to closely link to the tumour size, depth of invasion, lymphatic or venous invasion and UICC staging in line with the observation in breast carcinomas [19], indicating the inhibitory effects of parafibromin on tumour growth, invasion, metastasis and progression of gastric carcinomas. Drosophila Hyrax and its human orthologue, parafibromin, are required for nuclear transduction of the Wnt/Wg signal and bind directly to the C-terminal region of beta-catenin–Armadillo, thereby controlling transcriptional initiation and elongation by RNA polymerase II [15]. Parafibromin overexpression can inhibit colony formation, anchorage-dependent cell growth and cellular proliferation and induce cell cycle arrest in the G1 phase [28]. These findings demonstrated that loss of parafibromin expression had impact on the pathogenesis and progression of malignancies by promoting cellular proliferation. Additionally, parafibromin was expressed with a higher incidence in intestinal-type gastric cancer, which is presumed to arise from preceding dysplastic lesions, than diffuse-type one, which evolves without any precedent dysplastic changes. It is also demonstrated that distinct parafibromin expression underlies the molecular mechanisms for the differentiation of intestinal- and diffuse-type carcinomas.
Until now, there is yet no paper describing the prognostic significance of parafibromin expression in malignancies. Here, for the first time, we analysed the relation of parafibromin expression with the survival rate of 508 patients with gastric carcinoma. The results revealed a close link between its loss and worse survival. If stratified according to the depth of invasion, the significant link disappeared, indicating that the relationship between parafibromin expression and prognosis depends on the depth of invasion. The multivariate analysis demonstrated that age, depth of invasion, lymphatic invasion, lymph node metastasis, UICC staging and Lauren’s classification but not parafibromin expression, venous invasion or sex were independent prognostic factors for carcinomas. These findings suggested that parafibromin expression is a promising indicator for the favorable prognosis of gastric carcinoma patients, albeit not independent.
In the present study, a large number of gastric carcinoma cases were screened by TMA, which takes the advantages of high throughput, identical immunohistochemical conditions, and economy of samples, antibodies and time [33]. Although we used 2-mm-in-diametre needles, which are large enough to evaluate the morphological appearance and carefully selected representative regions with the reference of HE slides, it was difficult to avoid selection bias. Gill et al. [5] found that stronger parafibromin staining and more positive cells sometimes appeared at the edges of the parathyroid tumour than in the centre. This could be due to fixation methods, other processing issues or a biological phenomenon, for example tissue hypoxia in the centre of large tumours. Additionally, the collection of our samples (e.g. gastritis, adenoma and adenocarcinoma) respectively from the endoscopic biopsy, polypectomy, or surgical resection put forward their another possibility of selection bias because of different fixation and processing methods. Selvarajan et al. [19] found that parafibromin underexpression was correlated particularly with large tumour size which is in line with our finding. It was possible that weaker staining could be attributable to poor fixation properties in the centre of large tumours. Because tumour size is a key prognostic indicator, this artifact could explain the prognostic significance of parafibromin in gastric carcinomas. Therefore, it is a limitation of the present study not to separate the edge and centre of gastric carcinomas when establishing TMA. In the current study, the negatively staining carcinomas are associated with a negative internal control (stromal cells and lymphocytes) whereas the positive staining epithelium is adjacent to positive staining internal controls. Therefore, the negative staining of the carcinoma might be artificial, which should be considered as another limitation of the study. Our study might be mentioned as a preliminary experiment and the staining with original-size sections is an extensive work in the future using the gastric carcinoma samples, fixed and processed by the same approach.
In summary, downregulated parafibromin expression might play an important role in malignant transformation of gastric epithelial cells. Its reduced expression was closely related to growth, invasion, metastasis and worse prognosis of gastric carcinomas. Its expression could be employed to differentiate the intestinal- and diffuse-type carcinomas and underlay the molecular mechanism about the differentiation of both carcinomas. It was considered as a promising marker to indicate the pathobiological behaviours and prognosis of gastric carcinomas.

parafibromin
pathogenesis
prognosis
gastric carcinoma
progression


The UNESCO Universal Declaration on Bioethics and Human Rights: Perspectives from Kenya and South Africa

In October 2005, UNESCO (the United Nations Educational, Scientific and Cultural Organization) adopted the Universal Declaration on Bioethics and Human Rights. This was the culmination of nearly 2 years of deliberations and negotiations. As a non-binding instrument, the declaration must be incorporated by UNESCO’s member states into their national laws, regulations or policies in order to take effect. Based on documentary evidence and data from interviews, this paper compares the declaration’s universal principles with national bioethics guidelines and practice in Kenya and South Africa. It concentrates on areas of particular relevance to developing countries, such as protection of vulnerable persons and social responsibility. The comparison demonstrates the need for universal principles to be contextualised before they can be applied in a meaningful sense at national level. The paper also assesses the ‘added value’ of the declaration in terms of biomedical research ethics, given that there are already well-established international instruments on bioethics, namely the World Medical Association Declaration of Helsinki and the CIOMS (Council for International Organizations of Medical Sciences) guidelines on biomedical research. It may be that the added value lies as much in the follow-up capacity building activities being initiated by UNESCO as in the document itself.
Introduction
In October 2005,1 the United Nations Educational, Scientific and Cultural Organization (UNESCO) adopted the Universal Declaration on Bioethics and Human Rights. The preamble states: “It is necessary and timely for the international community to state universal principles that will provide a foundation for humanity’s response to the ever-increasing dilemmas and controversies that science and technology present for humankind and the environment” [20, p. 3]. As the declaration is non-binding, this universal foundation will be implemented at the level of the nation-state. The onus is on UNESCO’s member states to incorporate the declaration’s provisions into their national laws, regulations or policies. This paper examines the synergies between the declaration and bioethics regulation in two countries, Kenya and South Africa.
Before the UNESCO declaration was adopted, international instruments such as the World Medical Association (WMA) Declaration of Helsinki and the International Ethical Guidelines for Biomedical Research Involving Human Subjects of the Council for International Organizations of Medical Sciences (CIOMS) were already well-established in bioethics [5]. Indeed, the Helsinki declaration and the CIOMS guidelines are noted in the preamble of the UNESCO declaration [20]. These instruments have had a significant influence on the national bioethics policies of developing countries [1], including Kenya and South Africa.
The objectives of this paper are twofold. The first is to assess the translation of universal principles into national practice. The comparison between the UNESCO declaration and Kenyan and South African bioethics forms the basis for this analysis. The second is to locate the ‘added value’ of the declaration, particularly to developing countries, given the pre-existence of international level bioethics documents. Empirically based, the paper draws on documentary evidence and interviews conducted in Kenya and South Africa in 2005 and 2006.
Background
The empirical research for this paper was carried out as part of a larger doctoral project on the global governance of human genomic and biomedical research and in particular the part developing countries play in this. The theoretical framework for this project is provided by international relations, a sub-discipline of political science. While international relations theory is not referred to overtly in this paper’s analytical sections, it provides the context for the understanding of universality contained therein. That is, universality is explored pragmatically, with regard to the relationship between broad principles negotiated at international level and their subsequent adaptation to national level policy and practice, rather than philosophically, in terms of universal versus pluralist moral reasoning.
‘Bioethics’
What exactly is meant by ‘bioethics’ is notoriously difficult to determine. This is illustrated by the fact that the UNESCO declaration contains no clarification of the term. While a definition appeared in earlier drafts, the impossibility of reaching a consensus on wording necessitated its being left out of the final version [18]. For the purposes of this paper, bioethics is understood specifically in terms of the regulation of biomedical research. The UNESCO declaration itself has a wider remit, covering medical practice and protection of biodiversity and the environment as well as research ethics [20]. Since the overall doctoral project is concerned chiefly with human subjects research, however, these broader considerations will not be discussed here.
Methodology and Limitations
Much of contemporary international relations theory concerns the roles of both state and non-state actors in global governance mechanisms. Reflecting this, the doctoral fieldwork consisted of 70 semi-structured interviews with a range of persons considered to be stakeholders in genomics and bioethics in France, the United Kingdom, Kenya and South Africa.2 The sample was chosen to reflect different societal perspectives on genomics and bioethics and thus consisted of those who formulate policy at international and national levels, those who must implement these policies in laboratories and ethics committees, those who claim to represent public concerns and those with commercial interests. The breakdown of sectors was as follows: policy-makers (20), scientists (17), ethicists3 (18), civil society representatives (12) and businesspersons (3).4 Of these 70 interviews, the data used in this paper draw on only 22 and come mostly from those with members of research ethics committees. Thus they are illustrative rather than representative.
Studying a declaration as it evolves5 makes for exciting and contemporary research. It also carries limitations, however; in this instance, fieldwork may have been conducted too early to enable an assessment of the social and political impact of the UNESCO declaration in Kenya and South Africa. In the wider context of the doctoral research project, this limitation will be addressed by the inclusion of UNESCO’s previous declarations on genomics and genetics, which have had longer to become established within national policy frameworks.6 It is worth noting that the research process became by default an awareness-raising exercise, in that many of the people interviewed were previously unaware of the bioethics declaration, or indeed its predecessors.
Previous Studies
The UNESCO declaration features in several publications. Developing World Bioethics devoted a whole issue to the draft text in September 2005. The articles were largely critical, questioning the content of the draft declaration, how it had been put together and whether UNESCO was the right body to be taking on such an endeavour [8]. Professor Henk ten Have, Director of the Division of Ethics of Science and Technology at UNESCO, responded that the journal’s contributors were perhaps not au fait with how UN agencies work [14]. After the declaration was adopted, Herman Nys wrote an editorial for the European Journal of Health Law outlining its basic tenets and comparing it to the European Convention on Human Rights and Biomedicine. He emphasised the obligations of states to take on the declaration, despite its being legally non-binding [10]. Professor ten Have has himself written about the declaration, in the wider context of UNESCO’s activities in ethics. In his article in the Kennedy Institute of Ethics Journal he described how these activities fall into three areas, namely the adoption of normative instruments such as the declaration and their subsequent implementation through national level capacity building and awareness raising [6].
There has been a growing literature in recent years on research ethics in developing countries. A few examples, taken from the British Medical Journal, the Bulletin of the World Health Organization and PloS Medicine, will serve to illustrate that contextualisation of international instruments at national level and capacity for ethical review have been among the major concerns raised. Sylvester Chima has suggested that international guidelines need to be interpreted legislatively at local and regional levels and has thus called on the African Union to pass binding directives that are nevertheless adaptable to the laws of each state [3]. Kass et al., in a case study published in January 2007 reviewing the practices of research ethics committees in several African countries, including Kenya and South Africa, highlighted insufficient funding and training as the biggest challenges facing these committees and proposed that workshops be set up on how to apply ethical principles at local levels. They also called for more empirical investigation of ethics in African research [7]. Peter Singer and Solomon Benatar, in a 2001 article on the Helsinki declaration, contended that building capacity in research ethics will have far more impact on ethical standards than “revisions of this or any other research ethics code,” implying that declarations themselves are of limited use unless the capacity exists to implement them [15, p. 747). Zulfiqar Bhutta has also argued that strengthening local capacity in bioethics is key to promoting ethical health research in developing countries [1].
The UNESCO Declaration
The Universal Declaration on Bioethics and Human Rights was adopted “by acclamation” at the UNESCO General Conference on 19 October 2005 [20, p. 5). This was the end of a process that began with an invitation by the 2001 General Conference to the UNESCO International Bioethics Committee to report on the possibility of elaborating a universal instrument on bioethics. The Committee recommended that this instrument be declaratory in nature (that is, non-binding) and the drafting process was launched in January 2004 [17, 19]. Thus the actual negotiation period lasted under 2 years. The declaration is aimed primarily at states, but can also be implemented by “individuals, groups, communities, institutions and corporations, public and private” where appropriate [20, p. 6]. It covers a wide range of bioethical principles, several of which had already become customary in bioethics and feature in documents such as the Helsinki declaration and the CIOMS guidelines (informed consent, for example). Some of the principles of particular relevance to developing countries will be elaborated further below.
Bioethics in Kenya and South Africa
Kenya and South Africa were chosen as fieldwork destinations because of their significant involvement in genomics and bioethics at local, national, regional and international levels. Both countries have recently adopted national guidelines on bioethics: in Kenya the 20047 National Council for Science and Technology (NCST) Guidelines for Ethical Conduct of Biomedical Research Involving Human Subjects in Kenya (‘human subjects guidelines’) and the 2005 Ministry of Health (MoH) Kenya National Guidelines for Research and Development of HIV/AIDS Vaccines (‘vaccines guidelines’) and in South Africa the Department of Health (DoH) Ethics in Health Research: Principles, Structures and Processes, which were drawn up by members of both the Department and the Interim National Health Research Ethics Committee, appointed under the National Health Act of 2003 [13].
Both Kenya and South Africa decided that national bioethics guidelines were necessary partly in order to protect poor and marginalised people from being exploited by unscrupulous researchers [9, 13]. Among other texts, the guidelines draw on the Helsinki declaration, the CIOMS guidelines8 and several documents from the World Health Organization and the United States, but are tailored to their national contexts, with specific provisions addressing the vulnerabilities that may have enabled past abuses [9, 11, 13]. Participants from ethics committees cited a similar assortment of guidelines and regulations—international, regional and national—as influential, including some from Europe, the United Kingdom and Australia.
Synergies between the UNESCO Declaration and Bioethics in Kenya and South Africa
As the UNESCO declaration is non-binding, if its principles are to be applied universally they will necessarily have to be reflected in national level documents and systems. This section compares the main tenets of the declaration that are of special relevance for developing countries with Kenyan and South African bioethics policy and practice. The comparison has two purposes. The first is to illustrate how internationally determined, universal principles might be implemented at national levels in developing countries. The second is to show to what extent these principles were already reflected in national systems, before the adoption of the declaration.
Community Consent—Article 6
The UNESCO declaration states that for a research project on a group or community, agreement from representatives may be sought, in addition to that of the individual participants [20]. Community Advisory Boards facilitate this in some areas of Kenya (interviews, K_06:2005 and K_25:2005) and dialogue with community members through such boards is a requirement for HIV/AIDS vaccines research [11]. The South African guidelines stipulate community involvement and consultation for research involving ‘collectivities’, on issues such as ownership of data and distribution of benefits and harms [13].9
Vulnerability—Article 8
This article holds that “individuals and groups of special vulnerability should be protected and the personal integrity of such individuals respected” [20, p. 8]. The Kenyan human subjects guidelines give special instructions concerning research with underdeveloped communities, prisoners, married women in rural areas and pregnant or lactating women [9], while the vaccines guidelines state that the vulnerable and poor must be protected from exploitation [11]. The South African guidelines invite ethics committees to be “especially vigilant when considering research proposals involving vulnerable populations” [13, Preamble] and contain detailed provisions for research involving pregnant women, foetuses, prisoners and vulnerable communities [13].
Cultural Diversity and Pluralism—Article 9
Under the declaration these should be given “due regard” [20, p. 8]. Again, the national guidelines contain specific examples of what this might entail. The Kenyan human subjects guidelines, in the context of gaining informed consent from married women in rural communities, remind researchers that each of Kenya’s 42 tribes will have “unique sociocultural backgrounds” [9, p. 11], while the South African guidelines, in a section on indigenous medical systems, call on researchers to respect the cultures and traditional values of all communities [13]. The Kenyan vaccines guidelines are less detailed and, in a similar vein to the UNESCO declaration, simply require that research teams be sensitive to “sociocultural issues,” without specifying what these issues might be [11, p. 30].
Social Responsibility—Article 14
This article of the UNESCO declaration is considered particularly innovative [6]. It is perhaps not surprising, then, that it should prove more specific than any of the national guidelines as to how social responsibility could be implemented. Where the Kenyan and South African guidelines require research to be relevant to study populations and each country in general, by addressing either “health needs” (Kenya) [9, pp. 13, 16] or “broad health and development needs” (South Africa) [13, p. 3], the declaration states that scientific and technological progress should advance: access to healthcare and medicines; adequate nutrition and water; improved living conditions and environment; elimination of marginalisation and exclusion; and reductions in poverty and illiteracy [20].
Benefit Sharing—Article 15
The declaration is similarly specific with regard to the sharing of benefits, giving seven examples of what form this could take, including sustainable assistance to research participants and provision of new health products stemming from research [20]. It is the latter of these examples which features most prominently in the national guidelines. In Kenya, if research produces positive results, treatments should be available to participants [9]. Also, before HIV/AIDS vaccine research can take place, the “availability, affordability and accessibility” of its potential products must be considered [11, p. 30]. Research proposals in South Africa must indicate whether there is a reasonable likelihood that participants will benefit from the research and whether they will receive long-term therapy after the study [13]. More widely, research findings must “be translatable into mechanisms for improving the health status of South Africans” [13, p. 3]. In practice, ethics committees in both countries try to assess the extent to which participants will benefit from a research project (interviews, K_25:2005 and SA_19:2006), although they do not always find this a straightforward endeavour (interviews, SA_10:2006 and SA_35:2006).
Transnational Practices—Article 21
This article calls on states to combat illicit trafficking of “organs, tissues, samples, genetic resources and genetic-related materials” [20, p. 10]. Transfer of tissues overseas has been a particular concern in both Kenya and South Africa, as reflected in their guidelines. The Kenyan vaccines guidelines aim to eliminate the unauthorised transfer of research materials and to this end contain a sample Biological Material Transfer Agreement [11]. Under chapter 810 of the South African National Health Act, an appendix to the DoH guidelines, the Health Minister may regulate “the importation and exportation of tissue, human cells, blood, blood products or gametes” [12, pp. 70, 62]. Ethics committees in both countries take tissue transfer equally seriously, examining closely any research protocol that involves movement of samples across borders. Generally they prefer a project to train people to analyse data in-country (interviews, K_17:2005, K_21:2005, K_25:2005, SA_19:2006, SA_21:2006 and SA_30:2006).
Bioethics Education, Training and Information—Article 23
Under this article states are invited to foster bioethics training and education “at all levels” and to encourage information dissemination on bioethics [20, p. 11]. Such training and education is perhaps more widespread in South Africa than Kenya, although the UNESCO National Commission was in the process of setting up a bioethics centre to serve the East Africa region at the time of interview (interviews, K_01:2005 and K_16:2005). Several South African universities run courses in ethics or bioethics and two training initiatives, IRENSA and SARETI, serve sub-Saharan Africa as a whole (including Kenya).11 All three set of guidelines advocate bioethics training [9, 11, 13].
Universal Principles in the National Context
The South African guidelines read:
The challenge to international research ethics is the development of universal rules for research at a time when health care is being delivered within very different health care systems and in a multicultural world in which people live under radically different economic conditions. [13, p. 7]This paper asks whether the UNESCO declaration has met this challenge. Although promulgated with the expressed purpose of stating universal principles, the declaration acknowledges that such cultural and economic differences exist, through its articles on community consent, cultural diversity, vulnerability, social responsibility and benefit sharing. The Kenyan and South African guidelines demonstrate how the declaration might be applied at state level, with their prescriptions concerning, for example, how researchers should engage with communities and which particular members of society should receive special attention as vulnerable persons. That states may need to adopt particular interpretations of the declaration’s principles in order to realise them in national and local contexts is highlighted by ten Have in his paper on UNESCO’s ethical activities. He describes the adoption of the declaration as only the “start of a long process of detailed elaboration and consequent application” [6, p. 342] and in the context of the articles on consent and social responsibility states:As principles they are universally adopted, but in practice their application must be tailored in multiple ways to accommodate different types of research and health care, categories of patients and problems, and cultural settings and traditions. [6, pp. 342–343]The need for any national implementation of the UNESCO declaration to be adequately contextualised is brought out in the interview data from Kenya and South Africa, as are differences in opinion as to the usefulness of international guidelines in general. In Kenya, an academic scientist and advisor to both the government and UNESCO stated that it would be necessary to work with those government officers responsible for effecting international documents in law, in order to “translate it [the declaration] into what is happening locally” (interview, K_13:2005). Similarly, the head of a research institution attested: Something which became more and more legalistic in the detail and binding but which ignored local realities would be unhelpful. Something which tried to establish clear, agreed principles, which had been widely consulted, not just between member states, but with the kind of communities that are affected, would be very useful. (Interview, K_07:2005)In South Africa, one ethics committee member thought that, given different cultural contexts and sensitivities, declarations such as the UNESCO one would have to be formulated as generally as possible to enable universal applicability (interview, SA_08:2006). Another was of the view that national and local ethics guidelines are “the things to follow,” because what is applicable in one country may not be applicable in another (interview, SA_19:2006). Several saw at least some value in the UNESCO endeavour, but cautioned that universal principles must not be applied unthinkingly. Perhaps mirroring ten Have’s prediction that elaboration of the declaration will be a long process, they described how working out how to apply such principles in different contexts is often the most challenging aspect of implementing international instruments (interviews, SA_10:2006, SA_17:2006 and SA_24:2006).
Added Value of the UNESCO Declaration
The UNESCO declaration is considered to be of added value because it is the first intergovernmental instrument on bioethics. Ten Have describes the commitment by governments to an agreed set of principles on bioethics as the “innovative dimension” of the declaration [6, p. 342]. The Helsinki declaration and the CIOMS guidelines, by contrast, have been adopted by professional organisations (although CIOMS is in official relations with the World Health Organization). Where the former is officially directed at physicians or researchers, however, the latter, like the UNESCO declaration, are to be used in designing national policy on biomedical research ethics, particularly in developing countries [4, 21].
It may take time for the significance of the UNESCO declaration’s governmental backing to filter through to those who practise research ethics. An anonymous reviewer of this paper described the declaration as the “common denominator of global ethical thought,” but for many stakeholders the Declaration of Helsinki and the CIOMS guidelines fulfil this role, their professional origins notwithstanding. The Helsinki declaration is generally considered the foremost document globally on medical research ethics [2, 5] and, with the CIOMS guidelines, forms the bedrock of research ethics in many developing countries [1]. In Kenya and South Africa, where policy-makers and ethics committees have looked to these two documents for guidance at the global level, several of those interviewed were unconvinced that the UNESCO declaration, which they saw as simply another international instrument on bioethics, was necessary. As the declaration was adopted only relatively recently, such perceptions may change as both its contents and its intergovernmental status become more widely established (particularly as it construes bioethics in broader terms than only medical research ethics).
If the UNESCO declaration is not to “remain paperwork,” as a non-binding instrument it must be effected by states [6, p. 343]. Kenya and South Africa already have national bioethics guidelines that complement the declaration to a large degree. They also have research ethics committees at institutional and national levels, or plans for constituting them. For those countries which have not already established bioethics systems, however, the UNESCO declaration could act as a catalyst to galvanise them into doing so. To this end, UNESCO’s ethics programme supports states in building bioethics capacity, through various activities. The first is to construct a database with information on ethics experts, institutions, teaching programmes and policies around the world. The second is to promote bioethics education, through teacher training and curriculum development. The third is to support the setting up of ethics committees and their subsequent operations [6]. These activities were broadly welcomed among those interviewed in both countries, with the caveat that they should not overlap too far with the initiatives of other organisations. UNESCO is in fact piloting an ethics teacher training course in Kenya in July 2007 [16], where there are fewer bioethics courses available than in South Africa.
One area where ethics committees in Kenya and South Africa are in need of support is in the monitoring of research projects once they have been approved, as required by all three sets of national guidelines [9, 11, 13]. In practice, as found by Kass et al., funding can be an issue. One prominent Kenyan ethics committee had only recently carried out its first on-the-spot inspection at the time when interview data were obtained in November 2005. Prevented from conducting these inspections more often by financial constraints, the committee generally relies on reports from investigators and word of mouth (interviews, K_17:2005 and K_25:2005). The South African DoH guidelines require at a minimum that ethics committees request annual reports from principal investigators and establish a complaints procedure [13]; members of two different ethics committees described such measures as “passive monitoring”. As in Kenya, committees do not have the resources to carry out site visits (interviews, SA_10:2006 and SA_17:2006). The UNESCO declaration itself does not offer much by way of assistance, the only article that could possibly be taken to relate to post-approval monitoring stating, “Appropriate assessment and adequate management of risk related to medicine, life sciences and associated technologies should be promoted” [20, p. 10].12 Perhaps UNESCO’s ethics programme could help with training in these areas or encourage better funding, however. As highlighted by Singer and Benatar, capacity building is likely to advance research ethics further than are reformulations of bioethical principles. It may be, then, that the added value of the declaration will prove to lie more in UNESCO’s follow-up activities than in the document itself, at least in the medium term, the innovative sections on social responsibility and benefit sharing notwithstanding.
Conclusion
This paper has revealed something of a loose consensus, at least between UNESCO and those involved in bioethics in Kenya and South Africa, on two counts. With regard to universal principles, any attempt to implement them at national levels must be contextualised. Working out how to apply such principles in particular social and economic contexts is arguably as challenging as reaching agreement on how they should be constituted in the first place. With regard to the usefulness of the UNESCO declaration, the significance of its adoption as the first intergovernmental instrument on bioethics must be matched by action in the form of capacity building for it to be of added value in the realm of biomedical research ethics.
The scope of this paper has been limited to a primarily pragmatic analysis of how universal principles can be applied at national level. The paper has not commented on whether the value of the universality that UNESCO has aimed towards in terms of a foundation for humanity is compromised if these applications are very different. Deeper reflection on the nature of universality in this context would require the input of trained bioethicists and philosophers. This paper highlights, then, the need for a cross-disciplinary approach to the analysis of international bioethics instruments. The scope of the study that engendered this paper was necessarily limited by the time and resource constraints of doctoral research. Further research could explore whether the experiences of other African countries have been similar to those of Kenya and South Africa. Francophone and lusophone states, in particular, may have quite different bioethical traditions and thus have had very different experiences in implementing bioethics policies.

unesco
kenya
south africa
universal principles
national bioethics
global bioethics