Advanced Trauma Life Support®. ABCDE from a radiological point of view

Accidents are the primary cause of death in patients aged 45 years or younger. In many countries, Advanced Trauma Life Support® (ATLS®) is the foundation on which trauma care is based. We will summarize the principles and the radiological aspects of the ATLS®, and we will discuss discrepancies with day to day practice and the radiological literature. Because the ATLS® is neither thorough nor up-to-date concerning several parts of radiology in trauma, it should not be adopted without serious attention to defining the indications and limitations pertaining to diagnostic imaging.
Introduction
In many countries, trauma care is based on Advanced Trauma Life Support® (ATLS®) [1]. Although the ATLS® manual and course are neither evidence based nor up-to-date concerning several parts of radiology in trauma, surgeons use the ATLS® recommendations, if present, routinely to support indications for diagnostic imaging. In addition, surgeons refer to the ATLS® unjustly with indications for imaging that are not supported by the ATLS® at all. Radiologists must be aware of this to intervene appropriately when sub-optimal imaging indications are presented. In this respect, knowing the content and the language of the ATLS can be helpful.
The objective of this review is to familiarize radiologist with the ATLS®. For this purpose, the rationale and indications of diagnostic imaging is assessed where it pertains to the ATLS® protocol. Instances of disagreement with the evidence in the literature and daily practice are highlighted [1].
Purpose of ATLS®
Accidents are the primary cause of death in patients aged 45 years or younger. In The Netherlands, 22 out of 100,000 people die each year because of accidental injury. For every one patient who dies, there are three survivors with serious disabilities [1, 2].
The purpose of adequate trauma care is to decrease this morbidity and mortality, which is expected to be achieved by fast, systematic, and effective assessment and treatment of the injured patient. Contrary to the ATLS guidelines, we think that imaging should play a prominent role in this process.
History of ATLS®
In 1976, an airplane with an orthopedic surgeon, his wife and children crashed in a corn field in Nebraska. The wife died. The surgeon and three of his four children were seriously injured. Although they survived, he considered the standard of care in the local hospital insufficient and decided to develop a system to improve the care for trauma victims, and thus, ATLS® was born.
Since the first ATLS® course in 1978, the concept has matured, has been disseminated around the world and has become the standard of emergency care in trauma patients in 46 countries [1].
The ATLS® concept is also used in the pre-hospital phase of trauma patient care and has been adopted for non-trauma medical emergencies and implemented in resuscitation protocols around the world.
Originally, ATLS® was designed for emergency situations where only one doctor and one nurse are present. Nowadays, ATLS® is also accepted as the standard of care for the first (golden) hour in level-1 trauma centers. The priorities of emergency trauma care according to the ATLS® principles are independent of the number of people caring for the patient.
ATLS® course
The ATLS® course is organized under license of the American College of Surgeons. Before the course, the students peruse the course manual. During a 2-day course, 16 students, mostly residents in surgery and anesthesiology, are trained by eight instructors. These instructors now number more than 100 in the Dutch ATLS® section, mostly surgeons and anesthesiologists but also two radiologists.
During the course, all emergency measures are taught and reviewed. By means of observing, practicing, and repeating the ATLS® concepts, the object of the course is that the students are capable to perform the necessary measures independently with the correct priorities.
The course concludes with a written and practical examination, which has a pass rate of 80–90%.
During the course, attention is also given to the multidisciplinary character of trauma care and the organization and logistics of trauma care in hospitals and surrounding area.
Radiology has a minor part in the course There are only 50 min to teach the systematic evaluation of chest radiographs and another 50 min to teach in cervical spine radiographs, with the objective for the student to be able to identify life-threatening and potentially life-threatening injuries on chest radiographs and identify fractures on the radiographs of the spine. There is no lecture or skill set concerning computed tomography (CT).
Essentials of ATLS®
ATLS® is a method to establish priorities in emergency trauma care. There are three underlying premises. (1) Treat the greatest threat to life first. (2) Indicated treatment must be applied even when a definitive diagnosis is not yet established. (3) A detailed history is not necessary to begin evaluation and treatment.
Therefore, the assessment of a trauma patient is divided in a primary and a secondary survey. In the primary survey, life-threatening injuries are diagnosed and treated simultaneously. All other injuries are evaluated in the secondary survey.
Primary survey
In the primary survey, the mnemonic ABCDE is used to remember the order of assessment with the purpose to treat first that kills first (Table 1). Airway obstruction kills quicker than difficulty of breathing caused by a pneumothorax, and a patient dies faster from bleeding from a splenic laceration then from a subdural hematoma.
Table 1In the primary survey, the mnemonic ABCDE is used to remember the order of assessment with the purpose to treat first that kills firstThe ABCDEAAirway and C-spine stabilizationBBreathingCCirculationDDisabilityEEnvironment and Exposure
Injuries are diagnosed and treated according to the ABCDE sequence. Only when abnormalities belonging to a letter are evaluated and treated as efficacious as possible can one continue with the next letter. In case of deterioration of a patient’s condition during assessment, one should return to ‘A.’ Imaging should not intervene with or postpone treatment.
A: Airway
The airway is the first priority in trauma care. All patients get 100% oxygen through a non-rebreathing mask. The airway is not compromised when the patient talks normally. A hoarse voice or audible breathing is suspicious; facial fractures and soft tissue injury of the neck can compromise the airway, while patients in a coma are not capable of keeping their airway patent. Endotracheal intubation is the most definite way to secure the airway.
In ‘A,’ the cervical spine needs to be immobilized. As long as the cervical spine is not cleared by physical examination, with or without diagnostic imaging, the spine should remain stabilized.
For the evaluation of ‘A,’ no diagnostic imaging is necessary. Imaging of the cervical spine is just an adjunct to the primary survey and not part of the ‘A,’ specifically because, as long as the spine is immobilized, possible spinal injury is stabilized and diagnostic imaging can be postponed. When ‘A’ is secure, one can continue with ‘B.’
B: Breathing
Breathing is the second item to be evaluated in trauma care.
Tension pneumothorax, massive hemothorax, flail thorax accompanied by pulmonary contusion, and an open pneumothorax compromise breathing acutely and can be diagnosed with physical examination alone and should be treated immediately. Most clinical problems in ‘B’ can be treated with relatively simple measures as endotracheal intubation, mechanical ventilation, needle thoracocentesis, or tube thoracostomy. The lack of a definitive diagnosis should never delay an indicated treatment. To evaluate the efficiency of breathing, a pulse oximeter can be applied.
Injuries, like a simple pneumothorax or hemothorax, rib fractures, and pulmonary contusion, are often more difficult to appreciate with physical examination. Because these conditions have less effect on the clinical condition of the patient, they can be identified in the secondary survey.
A chest radiograph is an adjunct to the primary survey and can be helpful in evaluating breathing difficulties and is necessary to evaluate the position of tubes and lines. When ‘B’ is stabilized, one can continue with ‘C.’
C: Circulation
Circulation is the third priority in the primary survey. Circulatory problems in trauma patients are usually caused by hemorrhage. The first action should be to stop the bleeding. Hemorrhage can be external from extremity and facial injury or not visible from bleeding in chest, abdomen, and pelvis. Instable pelvic fractures can be temporarily stabilized with a pelvic band to decrease blood loss.
Blood pressure and heart rate are measured; two intravenous lines are started, and blood is obtained for laboratory investigation.
In the search for internal blood loss, imaging can be very helpful. Radiological investigations such as a chest radiograph, when not already performed, ultrasound of the abdomen (focussed abdominal sonography in trauma, FAST) and a pelvic X-ray can suggest the localization of the bleeding.
A tension pneumothorax can be the cause of circulatory distress but is usually diagnosed and treated in ‘B.’ When a patient’s condition deteriorates, this diagnosis must be reconsidered. Hemodynamic instability can, infrequently, be caused by pericardial tamponade. Therefore, ultrasonography of the pericardial sac is part of a FAST examination. Other less frequently occurring causes of circulatory problems in trauma patients are myocardial contusion and loss of sympathetic tone caused by cervical and upper thoracic spinal cord injuries.
When it is not possible to stabilize the patient in the trauma suite, other intervention like operation or embolization should be performed. The remainder of the primary survey will be finished thereafter. When ‘C’ is stabilized, one can continue with ‘D.’
D: Disability
Disability should be assessed as the fourth priority in the primary survey, and this includes assessment of the neurological status. The Glasgow coma score (GCS) is used to evaluate the severity of head injury. This score is arrived at by scoring eye opening, best motor response, and best verbal response. Patients who open their eyes spontaneously, obey commands, and are normally oriented score a total of 15 points. The worst score is 3 points. A decreased GCS can be caused by a focal brain injury, such as an epidural hematoma, a subdural hematoma, or a cerebral contusion, and by diffuse brain injuries ranging from a mild contusion to diffuse axonal injury. To prevent secondary injury to the brain, optimal oxygenation and circulation are important. Also, impaired consciousness can be caused or aggravated by hypoxia or hypotension for which ABC stabilization is essential.
If a cranial CT is indicated, it should be done in the secondary survey.
E: Environment and exposure
Environment and exposure represent hypothermia, burns, and possible exposure to chemical and radioactive substances and should be evaluated and treated as the fifth priority in the primary survey.
At the end of the primary survey, before continuing with the secondary survey, the ABCDEs should be re-evaluated and confirmed.
Secondary survey
During the secondary survey, the patient is examined from head to toe, and appropriate additional radiographs of the thoracic and lumbar spine and the extremities are performed when indicated. CT scans, when indicated, are also done in the secondary survey.
If, during the secondary survey, the patient’s condition deteriorates, the primary survey should be repeated beginning with ‘A.’
The rigid spine board should be removed as early as possible because it is a serious risk for decubitus ulcer formation. Removing the hard backboard should not be delayed for the lone purpose of obtaining definitive spine radiographs.
Diagnostic imaging
Radiographs of the chest, pelvis, C-spine, and FAST are adjuncts to the primary survey.
Imaging is considered helpful but should be used judiciously and should not interrupt or delay the resuscitation process. When appropriate, radiography may be postponed until the secondary survey.
CT, contrast studies, and radiographs of the thoracic spine, lumbar spine, and extremities are also adjuncts to the secondary survey.
Imaging is most useful and efficient if consulting with a radiologist becomes routine [3, 4]. We extrapolate this advice to the trauma setting and endorse consultation with clinicians strongly; however, consulting a radiologist is not mentioned once in the ATLS® manual!
Contrary to (ever-increasing) daily practice, CT plays a minor role in the ATLS®. With the increasing use of CT in the evaluation of trauma patients, radiation exposure should be a major issue in the field of emergency radiology. CT scanners using an automatic exposure control technique can help to reduce radiation dose [5, 6].
Blunt trauma
Thorax
A chest radiograph must be obtained to document the position of tubes and lines and to evaluate for pneumothorax or hemothorax and mediastinal abnormalities. When not obtained in the primary survey, it should be done in the secondary survey. From the ATLS® manual, it is not clear if a chest radiograph should be performed in every patient [1]. However, this is in accordance with the literature. At present, no clinical decision rule is available concerning the indication for chest radiography in trauma patients.
A CT of the chest is considered an accurate screening method for traumatic aortic injury. If a contrast enhanced helical CT is negative for mediastinal hemorrhage and aortic injury, no additional diagnostic imaging is necessary [1].
If a CT is positive, the ATLS® manual states that the trauma surgeon is in the best position to determine which, if any other, diagnostic imaging is warranted. The possibility to construct multiplanar reconstructions (MPRs), maximum intensity projections (MIPs), volumetric, and virtual angioscopic three-dimensional views from MDCT data, making diagnostic angiography superfluous, is not stated [7]. The same post-processing tools can be used to differentiate between traumatic aortic injury and normal variants [7]. Neither consulting with a radiologist nor endovascular treatment of traumatic aortic injury are mentioned in the ATLS® manual.
Although it is recognized that the severity of pulmonary contusions does not correlate very well with the chest radiograph, a CT for the evaluation of pulmonary contusion is not mentioned. The superiority of CT in the detection of pneumothoraces and evaluation of the position of chest tubes is not stated [8, 9].
Abdomen
FAST is used in hemodynamic abnormal patients as a rapid, non-invasive, bedside, repeatable method to document fluid in the pericardial sac, hepato-renal fossa, spleno-renal fossa, and pelvis or pouch of Douglas. When FAST is available, it replaces diagnostic peritoneal lavage (DPL) [10]. FAST is a good performing screening tool in evaluating hypotensive trauma patients to differentiate those patients who do need urgent laparotomy from those who do not [11].
An abdominal CT is the most sensitive and specific investigation for the diagnosis of visceral and vascular injury; however, according to the ATLS®, an abdominal CT can only be performed in hemodynamically normal patients because a CT is considered time consuming. This is no longer true with the helical CT available today. The rate-limiting step has become the movement of the patient to the CT suite and on and off the CT table [12].
According to the ATLS® manual, an upper GI contrast study is the imaging method of choice in suspected diaphragm rupture. CT is not mentioned as an option. On the contrary, it is stated that CT misses diaphragmatic injuries. Although CT is not 100% sensitive, neither are GI contrast studies. A comparative study is not available. MDCT has the advantage that it is much easier and quicker to perform in trauma patients [1, 13]. Although no consensus of opinion exists, coronal, and sagittal multiplanar reconstructions (MPRs) might improve the accuracy of MDCT for the diagnosis of blunt traumatic diaphragm rupture [14, 15].
A final omission, and contrary to daily practice in many hospitals, is that interventional radiology is not mentioned as an adjunct to non-operative management in patients with abdominal visceral injury [16, 17].
Pelvis
It is recommended that a pelvic radiograph should be performed when the mechanism of injury or the physical examination indicates the possibility of a pelvic fracture.
Evaluation of the pelvis on an abdominal CT is not mentioned [18]. Compared to conventional radiography, CT has a higher sensitivity and specificity for the diagnosis of pelvic fractures, and MPRs can be used to delineate the full extend of the fracture [19, 20].
In a hemodynamically abnormal patient with a pelvic fracture and no indication for intra-abdominal hemorrhage on FAST or DPL, angiography with embolization is advised preceding surgical pelvic fixation.
In patients with an unstable pelvic fracture, inability to void, blood at the meatus, a scrotal hematoma, perineal ecchymoses, or a high-riding prostate, there is a suspicion of a urethral tear, and in these patients, a retrograde urethrogram should be performed before inserting a urinary catheter [1].
To exclude an intraperitoneal or extraperitoneal bladder rupture in patients with hematuria, a conventional or a CT cystogram can be performed [1, 21].
Cervical spine
Cervical spine radiographs are not indicated in patients who are awake, alert, sober, neurologically normal, have no neck pain or midline tenderness, can voluntary move their neck from side to side, and flex and extend without pain. In all other patients, a lateral, AP, and open-mouth odontoid view should be obtained. Although it is not mentioned in the ATLS® manual, this seems to be a combination of the Canadian C-spine rules and the Nexus criteria but the criterion ‘painful distracted injury’ has disappeared between the sixth and the seventh edition of the ATLS manual [1, 22–24]. Possibly, this was done because the definition of painful distracting injury is difficult, but if omitted, this reduces the sensitivity of the clinical decision rule [25].
On the lateral view of the cervical spine film, the base of the skull to the first thoracic vertebra must be assessed. If not all seven cervical vertebrae are visualized, a swimmers view must be obtained and is considered sufficient and safe [1]. Supine oblique views and, contrary to available literature, performing a CT scan of this area is not mentioned [26–28]. Further, of all suspicious areas and all not adequately visualized areas, an axial CT with 3-mm intervals should be obtained. In the cervical spine section, multidetector CT assessment with coronal and sagittal MPRs is not mentioned at all [1].
Performing a CT of the cervical spine without a preceding conventional radiograph as the screening method of choice is not mentioned. The recent ACR appropriateness criteria suggest otherwise [29].
To detect occult instability in patients without an altered level of consciousness, or those who complain of neck pain, flexion-extension radiographs of the C-spine may be obtained [1]. As flexion-extension radiographs are often non-diagnostic and necessitate movement of the spine that is potentially dangerous, at least, performing a CT first to exclude osseous injury or a magnetic resonance imaging (MRI) for the detection of ligamentous injury should be recommended today [27, 29, 30].
MRI is recommended in patients with neurological deficits to detect an epidural hematoma or a traumatic herniated disc. Contrary to the ACR appropriateness criteria, the ATLS states that, when a MRI is not available, CT myelography may be used [1, 29].
Angiography or CT angiography for the evaluation of injury to the carotid or vertebral artery is not mentioned [1].
Head
Again, according to the protocol, a cranial CT should be considered in all head-injured patients with a focal neurologic deficit of which the cause can be localized in the brain, a Glasgow coma scale less than 15, amnesia, loss of consciousness of more than 5 min, or severe headaches. This is insufficient to detect all clinical relevant brain injury. There is no reference to evidence-based clinical decision rules such as the Canadian head CT rule, the New Orleans head CT rule or the CHIP prediction rule [1, 31–34].
Thoracic and lumbar spine
The indications for diagnostic imaging are the same as for the cervical spine. AP and lateral radiographs should be performed with additional CT of suspicious areas [1].
It is not mentioned that the thoracic and lumbar spine can be reliably evaluated on a CT of thorax and abdomen. When a CT of thorax and abdomen has already, or will be, performed, conventional radiography does not have any additional value especially when MPRs of the spine are obtained [35].
Penetrating trauma
Chest
Pneumothorax and hemothorax can be diagnosed with a chest radiograph. Even in patients with a normal chest radiograph, a CT is advocated for the evaluation of heart, pericardium, and great vessels in patients with a suspicion of mediastinum transversing injury. For the heart and pericardial sac, a CT can be replaced by ultrasound, and for the major vessels, an angiography can be performed. For the evaluation of oesophageal injury, esophagography using a water-soluble contrast agent and complementary esophagoscopy should be performed. The trachea and bronchial tree can be evaluated by bronchoscopy.
Patients with penetrating injury of the lower chest below the transnipple line anterior and the inferior tip of the scapula posterior are considered to have abdominal trauma as well until proven otherwise [1].
Abdomen
A hemodynamically abnormal patient with a penetrating abdominal wound does not need diagnostic imaging but should undergo laparotomy immediately.
In a hemodynamically normal patient, an upright chest radiograph can document intraperitoneal air and is useful to exclude hemothorax or pneumothorax. An abdominal radiograph (supine, upright, or lateral decubitus) may be useful in hemodynamically normal patients to detect extra-luminal air in the retroperitoneum or free air under the diaphragm.
In all patients with penetrating abdominal injury, an emergency laparotomy is a reasonable option, especially in patients with gunshot wounds. In initially asymptomatic patients with a lower chest wound or injuries to the back or flank, the ATLS® considers double or triple contrast CT, DPL, and serial physical examination less invasive diagnostic options, equivalent to each other [1].
For asymptomatic patients with anterior abdominal stab wounds, DPL, laparoscopy, and serial physical examination are mentioned as diagnostic options. However, although there is a significant body of evidence that this may not be optimal, CT is not mentioned as a diagnostic option in these patients [36].
Conclusion
ATLS® is a well-tried systematic approach for the assessment of trauma patients. In multidisciplinary trauma care, it is beneficial and, maybe, even mandatory for effective communication that all members of the trauma team, including the radiologist, speak the same ATLS® language.
Although imaging should not intervene with or postpone treatment, a chest radiograph, pelvic radiograph, and FAST can direct treatment decisions and should be performed in the primary survey when indicated. Imaging of the cervical spine is also an adjunct to the primary survey but can be postponed as long as the spine is immobilized. All other imaging should be done in the secondary survey.
Unfortunately, according to the ATLS®, CT plays a minor role in the evaluation of trauma victims. In the ATLS®, chest CT is only mentioned for the diagnosis of traumatic aortic injury but, in our experience, chest CT is valuable for the evaluation of pulmonary contusions and hemothorax and pneumothorax. Nowadays, abdominal CT is less time consuming than the ATLS® states and can be used to evaluate the extent of the abdominal injury in patients in whom no immediate laparotomy is indicated to evaluate the possibilities for non-operative management with or without endovascular embolization. The indications for head CT according to the ATLS® are insufficient to diagnose all patients with significant head injury. CT of the cervical spine can be used as a primary investigating tool and not only as an adjunct to conventional radiography. When a CT of the chest and abdomen is indicated, the thoracic and lumbar spine, as well as the pelvis, can be evaluated on the axial CT images combined with coronal and sagittal multiplanar reconstructions, and in these cases, conventional radiography of the spine and pelvis do not have any additional diagnostic value.
Because the ATLS® is neither thorough nor up-to-date concerning several parts of radiology in trauma, it should not be adopted without questions to define indications for diagnostic imaging. Consultation between clinicians and radiologists can improve the efficiency and quality of diagnostic imaging in trauma patients.

trauma
radiology
diagnostic imaging
spine
thorax
abdomen


Nasal NK/T-cell lymphoma: epidemiology and pathogenesis

Nasal NK/T-cell lymphoma (NKTCL) is an uncommon disease, but usually shows a highly aggressive clinical course. The disease is much more frequent in Asian and Latin American countries than in Western countries, and is universally associated with Epstein–Barr virus (EBV) infection. Analyses of gene mutations, especially p53 and c-KIT, revealed the different frequencies by district. Epidemiological studies revealed the changes of the disease frequency in Korea during the period from 1977–1989 to 1990–1996. Case-control study showed that the exposure to pesticides and chemical solvents could be causative of NKTCL. Further studies including HLA antigen typing of patients is necessary to further clarify the disease mechanism.
Rapid destruction of the nose and face (midline) was firstly described by McBride in 1897 [1]. Macroscopically the lesions usually looked like necrotic granuloma and the patients showed aggressive and lethal course, therefore the term “lethal midline granuloma (LMG)” or “granuloma gangrenescens” were used for this condition. Later the term LMG is popularly used. It had became evident that the LMG is composed of three different types of histology, i.e. Wegener’s granulomatosis (WG), polymorphic reticulosis (PR) or midline malignant reticulosis (MMR), and malignant lymphoma [2, 3]. These diseases could be histologically differentiated from each other by taking clinical findings into account. WG is usually characterized by generalized necrotizing vasculitis involving both arteries and veins and the presence of glomerulitis [4]. Cancer and specific inflammation such as tuberculosis also cause a condition indistinguishable from LMG.
PR exhibits a polymorphous pattern of proliferation consisting of large atypical cells with mono- or multinucleus, small lymphocytes, plasma cells, benign-appearing macrophages, neutrophils and much less frequently eosinophils. PR had been considered as a variant of malignant lymphomas because the disease is frequently disseminated [2]. The nature of proliferating cells in PR had been controversial. Once the disease was termed as nasal T-cell lymphoma because the proliferating cells showed a positive immunoreactivity for polyclonal antibodies against T-cells [5, 6]. However, monoclonal rerrangement of the T-cell receptor genes was seldomly found in cases with “nasal T-cell lymphoma” [7]. Later Ng et al. [8] reported that the tumor cells showed a positive immunoreactivity for natural killer (NK) cell marker CD56. Subsequently it was shown that proliferating cells in PR had large granular lymphocyte morphology [9], which is a character of NK cells or cytotoxic T-lymphocytes. There have been accumulating evidences that PR is a neoplasm of activated NK cells [10–13]: the proliferating cells usually show CD2+ , CD56+ , CD3ε+, CD7−, CD16−, cytotoxic granule-associated proteins+ and do not exhibit rearrangement of T-cell receptor or immunoglobulin genes (Fig. 1). Meanwhile rearrangement of T-cell receptor genes was recorded in the rare cases of PR [14, 15]. Although the upper respiratory tract, especially nasal region, is the common site of presentation, NK/T-cell lymphoma of nasal type may present in diverse extranodal sites such as gastrointestinal tract, skin, testis, liver, and spleen [16]. In the World Health Organization classification, NK/T-cell lymphoma encompasses the lymphomas involving nasal area and nonnasal area. Until present, there have been many review articles on the clinical, pathological, immunohistochemical, and immunogenetical aspects of NK/T-cell lymphomas involving upper respiratory tract. This review focuses on the epidemiology and molecular pathogenesis of nasal NK/T-cell lymphoma (NLTCL).
Fig. 1HE. Polymorphous pattern of proliferation in the nasal cavity. Large cells show positive immunoreactivity with CD3ε, TIA-1, and CD56. ABC method, ×400
Association with Epstein–Barr virus (EBV)
The association between EBV and human malignancies, including endemic Burkitt’s lymphoma, Hodgkin lymphoma and non-Hodgkin’s lymphoma (NHL) of either B- or T-immunophenotypes, has been reported. Etiological role of EBV for development of NKTCL was firstly reported in 1990 [17]. Subsequent study revealed the constant association of NKTCL with EBV in the world (Fig. 2) (Table 1) [18–23]. Sino-nasal lymphomas are immunophenotypically classified into NK/T-(CD56+), T-, and B-cell type with distinctive clinical features [24]. CD56 positivity, therefore NKTCL, was closely associated with EBV positivity among sino-nasal lymphomas [22]. EBV could be subtyped based on the difference in sequence of EBNA2 region, i.e. type A and type B [25]. Almost all of the NKTCL in Korea and Japan had type A EBV, which is identical with the previous literature reporting that most cases of EBV-associated malignancies of immunocompetent patients in Asia had type A EBV [26]. Predominance of type A EBV was also found in NKTCL in Malaysia [27], indicating the predominance of type A EBV in NKTCL in Asia. Several studies indicated the occurrence of type B EBV in lymphoma of immunocompromised patients [28]. Borisch et al. [29] reported that three of six cases of NKTCL in Switzerland had type B EBV, although no findings suggestive of immunodeficiency were found in these patients. These findings suggest a geographic difference in the distribution of EBV subtype in the NKTCL.
Fig. 2In situ hybridization with EBER-1 probe reveals positive signals in the nucleus of proliferating cell
Table 1EBV in nasal NK/T-cell lymphomaSourceCountryEBV positive rate (%)EBV subtype Expression of latent[18]a
France7/7 (100)NA7/7 LMP+[20]China21/21 (100)NA5/21 LMP-1+[21]Japan11/12 (92)10/11 typeA8/12 LMP-1+[22]Korea15/16 (94)15/15 typeA7/15 LMP-1+[24]Indonesia18/20 (90)NANAEBV Epstein–Barr virus, LMP latent membrane protein, NA not availableaFresh tissue samples were used in the analyses
As shown in Table 1, the proliferating cells in NKTCL frequently express latent membrane protein (LMP) as revealed by immunohistochemistry. Latent infection gene products of EBV, EBNA-2 and LMP-1, serve as target antigens for the elimination of EBV infected cells by host cytotoxic T-lymphocytes (CTL) [30]. Shen et al. [31] showed that NKTCL cells are able to provide target epitopes of EBV for CTL. In immunocompromised hosts, proliferating cells expressing EBNA-2 and LMP-1 can escape from immune surveillance by the host CTL, which might result in the development of malignant lymphomas [32]. However, systemic immunosuppression is not noted in patients with NKTCL, thus suggesting an unknown underlying mechanism for escape of LMP-1-expressing tumor cells from the CTL. Several mechanisms such as downregulation of the immunogenic EBV nuclear antigens and preferential selection of the deletion genotype of LMP-1 was reported by Chiang et al. [33, 34] and expression of IL-10, an immunosuppressive cytokine, was reported by Shen et al. [31]. The cells expressing viral antigens are eliminated primarily by CTL in a MHC-class-I-restricted manner [35]. Two CTL-epitopes were identified in LMP-1 that are possibly pan A*02-restricted [36]. It is possible that NKTCL patients show lower frequencies of A*02 allele compared with those in the normal population. Indeed high-resolution genetic typing revealed significantly lower frequency of HLA-A*0201 in NKTCL than in normal population [37]. These findings suggest that HLA-A*0201-restricted CTL responses may function in vivo to suppress the development of NKTCL, or in other words, role of EBV for NKTCL development.
Genetical changes
Lymphoma arises from clonal expansion of lymphoid cells that are transformed by the accumulation of genetic lesions affecting oncogenes and tumor suppressor genes. In general, amount of samples from NKTCL lesions available for genetical analyses is small, and samples usually contain massive necrotic areas. Therefore information for genetical changes in NKTCL has been relatively limited until present.
Alterations of tumor suppressor genes and oncogenes
Polymerase chain reaction (PCR)—single strand conformation polymorphism (SSCP) followed by direct sequence method was employed for analyses of gene alterations in NKTCL. The genes analyzed by this method were p53, k-ras, c-kit, and β-catenin on the NKTCL cases from Asian countries [38–42] and Mexico [43]. p53 is a well-known tumor suppressor gene that causes cells with damaged DNA to arrest at the G1 phase of cell cycle or stimulating expression of the BAX gene, the protein that promotes apoptosis [44]. In a wide variety of human cancers, p53 gene mutations have been detected mainly in exon 5 through 8 [45]. K-RAS, c-KIT, and β-catenin genes are oncogenes. Greenblatt et al. [46] identified 50 studies in which sequencing of the entire coding region of p53 had been reported. Of the 560 mutations reported in those papers, 87% were found in exons 5–8, and most of the others were in exon 4 (8%). In the studies for NKTCL in Asia, exons 4–8 or exons 5–8 were examined in one institute (Table 2). The frequency of p53 mutations was various by district: high in Japan and Indonesia and low in Mexico, Korea, and Shenyang. Shenyang situates at north China, which is adjacent to the Korean peninsula, suggesting that environmental and genetical factors might generate the differences in frequency. Transitions (G:C to A:T) were the predominant pattern of mutations except for Mexico, in which number of cases with mutations was only five [43], thus the data from Mexico seemed to be not conclusive. Predominance of transition mutations suggests that some “endogenous” mutagens act in lymphomagenesis. The transition pattern of p53 mutations is known to be more susceptible to spontaneous genetic instability than transversion. While genetic instability as revealed by widespread microsatellite instability was not found in the cases with NKTCL [47].
Table 2p53 mutations in nasal NK/T-cell lymphomaNumber of cases Exons examinedFrequency of mutations (%)Predominance of transition mutationAsia Japan [41]584–862%yes Korea [41]424–831%yes China Beijing, Chengdu [38]425–848%yes Shenyang [40]204–840%yes Indonesia [42]274–863%yesMexico [43]215–824%no
Quintanilla-Martinez et al. reported the association of p53 overexpression with poor prognosis, and p53 mutations with large cell morphology and advanced stage [43, 48]. However these findings were not confirmed by other studies. [15, 38]
The c-KIT proto-oncogene encodes a receptor tyrosine kinase, which is involved in normal hematopoiesis, gametogenesis, and melanogenesis via the c-kit receptor-ligand system. [49]. Because the development of acute leukemia or malignant lymphoma was reported in transgenic mice expressing KIT [8, 14, 50], NKTCL in Asian countries was examined for the c-kit gene mutations [41]. Frequency of c-kit mutations was significantly higher in China (Beijing, Chengdu) (10 of 14 cases: 71.4%) [39] than in Japan (9 of 58 cases: 15.5%) [41], Korea (5 of 42 cases:11.9%) [41], northeast China (Shenyang) (2 of 20 cases:10%) [40], and Indonesia (3 of 27 cases :11.1%) [42]. These findings suggest that location-specific differences in etiological factors cause specific mutations in c-kit gene.
FAS Gene Mutations
Fas (Apo-1/CD95) is a 45 kDa membrane protein belonging to the tumor necrosis factor receptor family, and mediates programmed cell death (apoptosis) through binding of FAS ligand (Fas L) [51]. Fas consists of 325 amino acids with a single transmembrane domain, including signal peptide. The 80-amino acid portion in the cytoplasm, designated as a death signaling domain, is essential for the apoptotic signal transduction. FAS gene mutations were reported in about 10% cases with sporadic non-Hodgkin’s lymphoma [52]. NKTCL frequently co-express FAS and FAS ligand (Fas L), but the tumor cells seldom undergo apoptosis. Some mechanisms for resistance to FAS/FAS L - induced apoptosis might work in the development of NKTCL, thus FAS gene mutations could be one of the mechanisms. Two reports support this notion: NKTCL cells in 9 of 15 (60%) cases [53] and 7 of 14 (50%) cases [54] showed mutations of FAS gene. Mouse T-cell lymphoma cells transfected with mutated FAS genes were resistant to apoptosis (Fig. 3, Fig. 4) [54], indicating the mutations to be loss of function mutation. These findings suggest that accumulation of lymphoid cells with FAS mutations provides a basis for the development of NKTCL.
Fig. 3Summary of the FAS gene mutations found in patients with nasal NKTCL. The shaded rectangles at the COOH-terminus of the protein represents the small peptide added because of the frameshift in the gene encoding FAS.TM: transmembrane domain
Fig. 4The mouse WR19L cell line expressing recombinant human FAS protein with (T1102C, A978G, 1095 ins A) or without (wild type) mutations were incubated with various concentrations of anti-FAS antibody at 37.0°C for 16 h. Clones expressing FAS receptor with any mutations (A978G, 1095 ins A, T1102C) were resistant to apoptosis induced by the anti-FAS antibody
Others
Various cytogenetic alterations have been reported, of which deletions of 6q are the common [55].
Epidemiological features
It had been reported that NKTCL seemed to be relatively common among non-Hodgkin’s lymphoma in Hong Kong [56–58]: malignant lymphomas affecting nose and nasopharyngeal region constituted 7.2% of extranodal lymphomas [56] and 45 of 70 cases with malignant lymphomas in the nose, nasopharynx, and larynx had PR morphology [57]. This disease is occasionally encountered in the hospitals in Japan [5, 59, 60]. When one of the authors (KA) visited the USA in 1987, he noticed that the disease was quite rare in the USA, but they had consultation cases from Peru. Because Japanese and a part of Peruvian belong to Mongolian ethnic group, it was postulated that the Mongolian group might be much more frequently affected by this disease. Then they started to examine the frequency of NKTCL in Japan, Korea (Seoul), and China (Shanghai) during the period from 1987 to 1993. The results are summarized in Table 3 [61–63], in which the frequency of each disease constituting LMG is shown as the frequency per 100,000 patients who visited the Ear–Nose–Throat (ENT) clinic in 37 university hospitals in Japan, Ryukyu University Hospital in Okinawa, Japan, Yonsei University Hospital in Seoul, and Shanghai Medical University Hospital. All of the histological sections were reviewed by one of the authors (KA). Frequency of PR ranged from 8 to 40.8. That in the Institute of Laryngology and Otology, London (1966–1987) was four, showing two to ten times higher frequencies of the PR in the east Asian countries [61]. The disease is rare in the USA [64] and Europe [65]. The patients with NKTCL seem to be clustered also in Latin American countries and Indonesia [66, 67]. Information from other parts of the world such as Africa, the Middle and Near East, and Rusia is helpful to elucidate the etiology of this disease.
Table 3Frequency of lethal midline granuloma in Japan, Korea (Seoul), and China (Shanghai)DiseaseNumber of patients (frequency per 100,000 ENT patients)Japan other than Okinawa (1965–1986)Okinawa (1973–1991)Seoul (1979–1989)Shanghai (1979–1990)Wegener’s granulomatosis64(4)1(3)0(0)1(4)Polymorphic reticulosis114(8)9(27.4)56(40.8)73(9.8)Malignant lymphoma82(6)11(33.5)15(10.9)54(7.2)Others42(3)1(3)6(4.4)0(0)Total302(21)22(69.9)77(56)128(17)
At the late 1990s, otorhinolaryngologists in both Korea and Japan had the impression that the frequency of PR appeared to be decreasing. Then the changes in frequency of PR with time among cases from Seoul and 59 university hospitals in Japan were examined [68]: the frequency rate of PR per 100,000 outpatients of ENT clinics in Seoul decreased from 40 to 20 between the periods of 1977–1989 and 1990–1996. However, there were no significant changes in Japan during the period studies.
Life-style and environmental factors
Epidemiological studies have revealed that the NKTCL occurs much more frequently in Asian countries than in Western countries and it is closely associated with EBV infection. There are differences in frequencies of p53 and c-kit gene mutations among patients with NKTCL in Japan, China, and Korea. Recently the first case of familial NKTCL affecting a father and one of his six children was reported [69]. They used large amounts of pesticide in a green house. An increase in the risk of developing NHL among individuals exposed to pesticides was reported [70, 71]. In addition, a correlation of exposure to certain pesticides and organochlorines with increased titers of antibodies to EBV was reported [72]. All these findings might suggest a causative role for some genetical, environmental and life style factors in the development of NKTCL. Therefore, the epidemiological study to elucidate whether socioenvironmental ambient factors contribute to the development of NKTCL was conducted as a collaborative study of Japan, Korea and China (Table 4) [73]. The odd ratio (OR) of NKTCL was 4.15 (95% confidence interval (CI), 1.74–9.37) for farmers, 2.81 (CI 1.49–5.29) for producers of crops, and 4.01 (CI 1.99–8.09) for pesticide users. The ORs for crop producers, who minimized their exposure to pesticides by using gloves and glasses, and sprinkling downwind at the time of pesticide use, were 3.30 (CI 1.28–8.54), 1.18 (CI 0.11–12.13), and 2.20 (CI 0.88–5.53), respectively, which were lower than those for producers who did not take these precautions. Exposures to pesticides and chemical solvents could be causative factors for NKTCL. Previous studies showed the increased risk of NHL in individuals using pesticides, especially phenoxyacetic acid-type herbicides [74]. Association of pesticides with risk of developing t(14;18) positive-NHL, but not t(14;18) negative-NHL, was reported [75].
Table 4Risk of nasal NK/T-cell lymphoma in relation to cultivation of crops and pesticide useOdds ration (OR)a
Number of cases N = 88Number of controls N = 30595% CILowerUpperCultivation of crops At present2.8127361.495.29 More than 5 years5.0824192.4710.43Pesticides Users4.0123231.998.09Type of pesticide Herbicide3.1713161.367.38 Insecticide3.4520211.677.13 Fungicide6.051061.9818.46Precautions Gloves used3.3010111.288.54 Gloves not used 4.7613121.9311.72 Mask used5.4414102.2013.47 Mask not used 2.829131.087.37 Glasses used1.18130.1112.13 Glasses not used4.5222202.179.42 Sprinkling downward attended2.209160.885.53 Sprinkling downward not attended8.451473.0123.70CI confidence intervalaAdjusted for age (<40, 40–59, >60), sex (male/female) and country (Japan, Korea and China)
Conclusions
Clinical course of patients with NKTCL is usually highly aggressive. Therefore clarification of risk factors for disease development is especially important to establish a strategy for disease prevention. Because EBV infection and pesticides could be risk factors for NKTCL, investigation on effects of pesticides for EBV activation is needed. Employment of the similar kind of the epidemiological study is desirable in other areas than East Asia. Patients with NKTCL cluster in Asia and Latin American countries, therefore some genetical factors might be involved in the disease development. Further studies including HLA antigen typing of patients is important to further clarify the mechanism for disease development.

epidemiology
epstein–barr virus
pesticide
p53 gene
c-kit gene
nasal lymphoma
nk/t-cell type


The 894G>T variant in the endothelial nitric oxide synthase gene and spina bifida risk

The 894G>T single nucleotide polymorphism (SNP) in the endothelial NOS (NOS3) gene, has recently been associated with embryonic spina bifida risk. In this study, a possible association between the NOS3 894G>T SNP and spina bifida risk in both mothers and children in a Dutch population was examined using both a case-control design and a transmission disequilibrium test (TDT). Possible interactions between the NOS3 894G>T SNP and the MTHFR 677C>T SNP, elevated plasma homocysteine, and decreased plasma folate concentrations were also studied. The NOS3 894TT genotype did not increase spina bifida risk in mothers or children (OR 1.50, 95%CI 0.71–3.19 and OR 1.78, 95%CI 0.75–4.25, respectively). The TDT demonstrated no preferential transmission of the NOS3 894T allele (Χ2 = 0.06, P = 0.81). In combination with the MTHFR 677TT genotype or elevated plasma homocysteine concentrations, the NOS3 894GT/TT genotype increased maternal spina bifida risk (OR 4.52, 95%CI 1.55–13.22 and OR 3.38, 95%CI 1.46–7.84, respectively). In our study population, the NOS3 894GT/TT genotype might be a risk factor for having a spina bifida affected child in mothers who already have an impaired homocysteine metabolism.
Introduction
Nitric oxide synthase (NOS) catalyzes the generation of nitric oxide (NO), an important signaling molecule that mediates many of its biological effects by activating the enzyme guanylyl cyclase, thereby increasing cyclic GMP synthesis (Murad 2006). Besides its important role in cardiovascular control, NO has been suggested to play a role in development (Lee and Juchau 1994). NO has been demonstrated to be present in the neural tube of chick embryos at the time of neurulation (Traister et al. 2002) and regulates the balance between mitosis and programmed cell death (Plachta et al. 2003). Other studies show that NO can inhibit methionine synthase (MTR) enzyme activity, thereby interfering with homocysteine remethylation (Danishpajooh et al. 2001) and proper neurulation (Nachmany et al. 2006; Weil et al. 2004).
The 894G>T single nucleotide polymorphism (SNP) in the endothelial-derived nitric oxide synthase (NOS3) gene, identified by Hingorani et al. (1999), was shown to be associated with elevated plasma homocysteine levels in nonsmokers with low serum folate concentrations (Brown et al. 2003). More recently, it was demonstrated that the NOS3 894GT genotype is an embryonic risk factor for spina bifida (OR 1.63, 95%CI 1.09–2.42) (Brown et al. 2004).
In this study, we examined the relation between the NOS3 894G>T SNP and spina bifida risk in both mothers and children using a case-control design and a transmission disequilibrium test (TDT). An effect of the NOS3 894G>T SNP on plasma homocysteine or plasma folate concentration was examined as well as possible interactions between the NOS3 894G>T SNP and the MTHFR 677C>T SNP, elevated plasma homocysteine, and decreased plasma folate concentrations.
Materials and methods
Study population
The study population has been described in detail before (van der Linden et al. 2006). Briefly, the study population was recruited in collaboration with the BOSK and was extended by a group from the Pediatric Neurology Department of the Radboud University Nijmegen Medical Center. The study group included 109 spina bifida patients (61 girls and 48 boys, mean age 16.4 ± 11.3), their mothers (n = 121, mean age 42.9 ± 10.9) and fathers (n = 103, mean age 44.9 ± 10.2).
The control group consisted of 500 volunteers recruited from a general practice in The Hague (den Heijer et al. 1995). In the present study we only included the 292 women from this control group (mean age 50.6 ± 13.5). The pediatric control group included 234 children (119 girls and 115 boys, mean age 8.4 ± 6.4) (van Beynum et al. 1999). The children of secondary school age were healthy volunteers, and the younger children were, for ethical reasons, recruited in a hospital setting. The local medical ethics committee approved the study.
For the present study, DNA of 102 spina bifida patients, 116 mothers, 101 fathers, 265 control women and 211 pediatric controls was available for genotyping.
Biochemical determinations
Plasma total homocysteine concentration was measured in EDTA plasma by HPLC and fluorescence detection, as previously described by te Poele-Pothoff et al. (1995).
Plasma folate concentration was determined using the Dualcount Solid Phase Boil Radio assay (Diagnostic Product Corporation, Los Angeles, CA).
PCR amplification and genotype determination
NOS3 894G>T genotyping was performed according to Hingorani et al. (1999). Primer annealing took place at a temperature of 58°C. The PCR product of 206 bp was cut with the restriction enzyme MboI, which cuts the T allele in fragments of 119 and 87 bp.
Statistical analyses
Case-control study
Odds ratios (ORs) and their 95% confidence intervals (95%CI) were calculated by means of logistic regression analysis.
The effect of the NOS3 894G>T SNP on plasma homocysteine and plasma folate concentration was studied by means of linear regression analysis. Since the distributions of plasma homocysteine and plasma folate concentrations are positively skewed, these variables were logarithmically transformed prior to linear regression analysis.
Deviation from Hardy–Weinberg equilibrium was calculated to assess the accuracy of the genotyping assay.
The power of the case-control study was calculated using the Genetic Power Calculator (Purcell et al. 2003). All tests were performed using SPSS software package version 12.0.1. Statistical significance was accepted at a two-tailed P < 0.05 or 95%CI of an odds ratio that did not include 1.0.
Transmission disequilibrium test
The relation between the NOS3 894G>T genotype of the spina bifida patients and spina bifida risk was assessed by evaluating the transmission of alleles from heterozygous parents to their affected offspring using the TDT, a family-based association test (Spielman et al. 1993). The power of the TDT was calculated using the Genetic Power Calculator (Purcell et al. 2003).
Results
Case-control study
The NOS3 894G>T genotype could be determined in 115 mothers of a spina bifida affected child and in 259 female controls. Genotype distribution did not differ from that expected under Hardy–Weinberg equilibrium (P = 0.83), and the power to detect a twofold increase in maternal spina bifida risk was 59%. Table 1 demonstrates that neither the NOS3 894GT genotype nor the NOS3 894TT genotype increased maternal spina bifida risk relative to the NOS3 894GG genotype. Since the effect of the NOS3 894GT genotype and the NOS3 894TT genotype on spina bifida risk was the same relative to the NOS3 894GG genotype, the NOS3 894GT and TT genotypes were combined to increase power, which resulted in a nonsignificant 36% increased risk of having a spina bifida affected child relative to the NOS3 894GG genotype (OR 1.36, 95%CI 0.87–2.11). The NOS3 894G>T SNP was not associated with plasma homocysteine or plasma folate concentrations in mothers and female controls (data not shown).
Table 1The NOS3 894G>T genotype distribution and unadjusted ORs in mothers of a spina bifida affected child and female controlsNOS3 894G>TMothers (%)Female controls (%)OR (95%CI)GG49 (42.6)130 (50.2)1aGT53 (46.1)106 (40.9)1.33 (0.83–2.11)TT13 (11.3)23 (8.9)1.50 (0.71–3.19)aReference group
In 95 spina bifida patients and 207 pediatric controls, NOS3 894G>T genotyping was performed successfully. The NOS3 894G>T genotype distribution did not differ from that expected under Hardy–Weinberg equilibrium (P = 0.76), and the power of the study was 51%. The NOS3 894GT genotype increased spina bifida risk in children whereas the NOS3 894TT genotype did not (Table 2). Combining the NOS3 894GT and 894TT genotypes resulted in a 66% increase in spina bifida risk relative to the NOS3 894GG genotype (OR 1.66, 95%CI 1.02–2.72). In the pediatric controls, the NOS3 894GT and 894TT genotypes were associated with increased plasma homocysteine concentrations relative to the NOS3 894GG genotype (14.7% increase, 95%CI 4.2–26.2% and 23.5% increase, 95%CI 3.9–46.8%, respectively). There was no association between the NOS3 894G>T SNP and plasma folate concentration in children (data not shown).
Table 2The NOS3 894G>T genotype distribution and unadjusted ORs in spina bifida patients and pediatric controlsNOS3 894G>TSB patients (%)Pediatric controls (%)OR (95%CI)GG39 (41.1)111 (53.6)1aGT46 (48.4)80 (38.6)1.64 (0.98–2.74)TT10 (10.5)16 (7.7)1.78 (0.75–4.25)aReference group
Transmission disequilibrium test
In this study there were 75 complete mother–father–child triads, and the power to detect a twofold increase in spina bifida risk was 32%. In 36 out of 70 informative transmissions (51.4%), the NOS3 894T allele was transmitted to the child, which resulted in a Χ2 of 0.06 (P = 0.81).
Gene-gene interaction
Data on both the NOS3 894G>T genotype and the MTHFR 677C>T genotype were present for 110 mothers of a spina bifida affected child and 259 control women. Since the MTHFR 677CT genotype did not increase spina bifida risk relative to the MTHFR 677CC genotype, we decided to combine the MTHFR 677CC and CT genotypes to increase power. In combination with the MTHFR 677TT genotype, the NOS3 894GT/TT genotype increased the risk of having spina bifida affected offspring 4.5 times relative to the NOS3 894GG genotype combined with the MTHFR 677CC/CT genotype (OR 4.52, 95%CI 1.55–13.22) (Table 3).
Table 3Maternal spina bifida risk for the combination of the NOS3 894G>T SNP and the MTHFR 677C>T SNPNOS3 894G>TMTHFR 677C>TMothers (%)Female controls (%)OR (95%CI)GGCC/CT42 (38.2)114 (44.0)1aGGTT6 (5.5)16 (6.2)1.02 (0.37–2.77)GT/TTCC/CT52 (47.3)123 (47.5)1.15 (0.71–1.85)GT/TTTT10 (9.1)6 (2.3)4.52 (1.55–13.22)aReference group
In children, there was no interaction between the NOS3 894GT/TT genotype and the MTHFR 677TT genotype (OR 1.23, 95%CI 0.31–4.91) (data not shown).
Gene-metabolite interactions
Since the MTHFR 677C>T SNP is a genetic determinant of plasma folate and plasma homocysteine concentrations, possible interactions between the NOS3 894G>T SNP and these metabolites were examined in mothers. In combination with decreased plasma folate concentrations (<8.52 nmol/L), the NOS3 894GT/TT genotype did not increase maternal spina bifida risk relative to the NOS3 894GG genotype in combination with normal plasma folate concentrations (≥8.52 nmol/L) (OR 1.14, 95%CI 0.49–2.67) (data not shown). The risk of having spina bifida affected offspring increased to 3.4-fold when the NOS3 894GT/TT genotype was present in combination with elevated plasma homocysteine concentrations (≥12.56 μmol/L) (OR 3.38, 95%CI 1.46–7.84) (Table 4).
Table 4Maternal spina bifida risk for the combination of the NOS3 894G>T SNP and elevated plasma homocysteine concentration, adjusted for ageNOS3 894G>TPlasma Hcy conc (μmol/L)aMothers (%)Female controls (%)OR (95%CI)GG<12.5642 (36.8)100 (38.6)1bGG≥12.567 (6.1)30 (11.6)0.66 (0.26–1.65)GT/TT<12.5647 (41.2)108 (41.7)1.17 (0.69–1.96)GT/TT≥12.5618 (15.8)21 (8.1)3.38 (1.46–7.84)aThe 80th percentile of the control plasma homocysteine distribution was used to define elevated plasma homocysteine concentrationsbReference group
Discussion
In this study, the NOS3 894TT genotype did not significantly increase spina bifida risk, and the TDT demonstrated no preferential transmission of the NOS3 894T allele. However, in combination with the MTHFR 677TT genotype or elevated plasma homocysteine concentrations, the NOS3 894GT/TT genotype increased maternal spina bifida risk 4.5- and 3.4-fold, respectively.
Using a likelihood ratio test (LRT) for both proband and mother trios, Brown et al. (2004) demonstrated the NOS3 894GT genotype, but not the NOS3 894TT genotype, to be an embryonic risk factor for spina bifida (OR 1.63, 95%CI 1.09–2.42). Data from our case-control study might be in line with this observation of Brown et al. (2004); however, based on the effect of the heterozygous NOS3 894GT genotype on spina bifida risk, a larger effect of the NOS3 894TT genotype on spina bifida risk would be expected. The observation that the NOS3 894GT genotype increases spina bifida risk and the NOS3 894TT genotype does not could be explained as a chance finding, but could also be the result of lack of power, as also suggested by Brown et al. (2004).
Despite the small numbers on which the analyses are based, the possible interactions between the NOS3 894GT/TT genotype and the MTHFR 677TT genotype or elevated plasma homocysteine concentrations are interesting and may suggest a role for NOS3 uncoupling or S-nitrosation. The NOS3 894G>T SNP (Glu298Asp) has been suggested to impair NOS3 protein function and/or activity resulting in decreased NO production (Sofowora et al. 2001; Tesauro et al. 2000), although results are contradictory (Fairchild et al. 2001). Elevated homocysteine concentrations have been demonstrated to switch the NOS3 enzyme to an uncoupled state directed to the synthesis of reactive oxygen species (ROS) rather than NO synthesis (Topal et al. 2004). The combination of the NOS3 894GT/TT genotype and elevated plasma homocysteine concentrations may lead to such a decrease in NO levels that neurulation is hampered. NO can also react with homocysteine to form S-nitrosothiols, thereby modulating homocysteine availability (Stamler et al. 1993). As previously suggested by our group (Heil et al. 2004), decreased NO levels may result in decreased S-nitrosothiol formation, resulting in less capturing of homocysteine into S-nitrosoHcy. The subsequent increase in plasma homocysteine concentrations might influence proper neural tube closure, especially when plasma homocysteine concentrations are already high.
The results of our study suggest that the NOS3 894G>T SNP is a spina bifida risk factor in the mother and not in the child. Our results furthermore indicate a role for the homocysteine metabolism in the detrimental effects asserted by the NOS3 894G>T SNP. More studies on the association between the NOS3 894G>T SNP and spina bifida in larger populations are warranted.

spina bifida
nos3 894g>t
transmission disequilibrium test


The microtubule-associated protein tau is phosphorylated by Syk

Aberrant phosphorylation of tau protein on serine and threonine residues has been shown to be critical in neurodegenerative disorders called tauopathies. An increasing amount of data suggest that tyrosine phosphorylation of tau might play an equally important role in pathology, with at least three putative tyrosine kinases of tau identified to date. It was recently shown that the tyrosine kinase Syk could efficiently phosphorylate α-synuclein, the aggregated protein found in Parkinson's disease and other synucleinopathies. We report herein that Syk is also a tau kinase, phosphorylating tau in vitro and in CHO cells when both proteins are expressed exogenously. In CHO cells, we have also demonstrated by co-immunoprecipitation that Syk binds to tau. Finally, by site-directed mutagenesis substituting the tyrosine residues of tau with phenylalanine, we established that tyrosine 18 was the primary residue in tau phosphorylated by Syk. The identification of Syk as a common tyrosine kinase of both tau and α-synuclein may be of potential significance in neurodegenerative disorders and also in neuronal physiology. These results bring another clue to the intriguing overlaps between tauopathies and synucleinopathies and provide new insights into the role of Syk in neuronal physiology.
Many neurodegenerative disorders are characterised by intracellular inclusions of highly insoluble proteins, and a classification based upon the main protein component of these aggregates is widely used. The term “synucleinopathies”, for instance, refers to a group of disorders including Parkinson's disease (PD) in which the synaptic protein α-synuclein (α-syn) forms neuronal or glial aggregates [1]. “Tauopathies” are another pathological entity in which the microtubule-associated protein tau self-assembles into filamentous inclusions called neurofibrillary tangles (NFT) [2]. The tauopathies include Alzheimer's disease (AD), progressive supranuclear palsy and corticobasal degeneration.
Human brain tau consists of a family of six isoforms, generated by the alternative splicing of a single mRNA transcript. Tau proteins contain either three (tau 3R) or four (tau 4R) tubulin-binding domains depending on the splicing of exon 10, and either one (tau 1N) two (tau 2N) or no (tau 0N) N-terminal inserts depending on the inclusion of exon 2, exons 2 and 3, or exclusion of both, respectively. Their apparent molecular weights range from 45 kDa for tau 0N3R isoform to 65 kDa for tau 2N4R. When aggregated in NFT, tau is abnormally hyperphosphorylated by a series of serine/threonine kinases, among which glycogen synthase kinase-3β, cyclin-dependent kinase 5 and casein kinase 1 appear to be the most relevant [3,4]. It has been hypothesized that this hyperphosphorylation contributes to neurodegeneration through the destabilisation of microtubules [5].
Recent data suggest that phosphorylation of tau also occurs on tyrosine residues and this could be of potential significance in pathological conditions [6,7]. Human tau protein possesses five tyrosine residues on positions 18, 29, 197, 310 and 394 (numbered according to the longest 2N4R tau isoform). Tau extracted from NFT of AD patients was found to be phosphorylated on tyrosine 18 (Y18) by immunocytochemistry using a phosphospecific antibody [6], and on tyrosine 394 (Y394) by mass spectrometry [8]. Tyrosine 197 (Y197) has been shown to be phosphorylated in tau aggregates from transgenic mice overexpressing mutant tau [9]. Recently, the dual specificity kinase tau-tubulin kinase 1 has been shown to phosphorylate tau in vitro on Y197 [10]. While Abl was recently described as a candidate tyrosine kinase for Y394 [8], Fyn is considered to be the kinase for Y18 [6].
The tyrosine kinase Syk is known for its critical role in signalling through immune receptors in leucocytes [11]. Syk tyrosine kinase expression is however not confined to hematopoietic cells, and has been reported in a variety of tissues [12]. Syk expression has been confirmed in the central nervous system (CNS) by immunoblot analysis of mouse brain homogenates, and its localization in mouse neuronal cytoplasm affirmed by immunohistochemistry [12]. While its function in the CNS remains unclear, Syk was recently found to be a kinase for α-syn [13]. The direct interaction of Syk and α-syn was proven by confocal microscopy and a dual-hybrid system approach [13]. The intriguing overlap in the pathophysiology of synucleinopathies and tauopathies prompted us to assess whether tau protein was also a substrate for Syk tyrosine kinase [14]. We herein report that Syk can phosphorylate Tau on Y18, and that the kinase and its substrate directly interact.
We first investigated whether Syk can directly phosphorylate tau in vitro. Recombinant human tau 2N4R was incubated with or without recombinant Syk or Abl [8]. Tau was phosphorylated in vitro by Syk and by its previously recognized tyrosine kinase Abl as judged on Western blots probed with P-Tyr-100 anti-phosphotyrosine antibody (Cell Signaling), whereas no phosphorylation was observed in experiments where tau was incubated without Syk (Fig. 1). In the presence of Syk, an additional band of approximately 70 kDa, migrating just above tau, was observed on the phosphotyrosine immunoblot. This band is likely to contain Syk since the kinase is known to autophosphorylate. Taken together, these results demonstrate that Syk can directly catalyze tau phosphorylation.
To determine whether Syk can phosphorylate tau in cells, co-transfection experiments were performed in CHO cells using Syk expression vectors together with untagged or V5-tagged tau. Cells were lysed and tau was immunoprecipitated with either anti-V5 antibody (Invitrogen) or with anti-tau antibody (Tau-5, BD Biosciences). Western analysis was then performed on immunoprecipitated tau using the anti-phosphotyrosine (P-Tyr-100, Cell Signaling), anti-tau (Tau-5) or anti-Syk (N-19, Santa Cruz) antibodies as previously described [8]. In preliminary experiments, a V5-tagged tau 2N4R construct and wild-type Syk were transiently coexpressed in CHO cells. We have previously shown that V5-tagged tau 2N4R migrates at approximately 70 kDa on SDS-PAGE [8]. Because of the close molecular weights of Syk (72 kDa) and the V5-tau construct, the two proteins co-migrated using conventional denaturing gel electrophoresis (data not shown). To better resolve the two proteins, we used two complementary approaches. In a first set of experiments, we expressed V5-tagged tau 2N4R along with a Syk-GFP construct that migrates at 100 kDa. The Syk-GFP construct was proven to remain functionally active in previous studies [15]. In a second set of experiments, we coexpressed wild-type untagged Syk along with the shortest tau isoform (0N3R), which migrates at 45 kDa. Tau protein was phosphorylated on tyrosine when co-transfected with Syk-GFP construct or with its known tyrosine kinase Abl (Fig. 2A). Additional positive controls included tau-transfected cells treated with the tyrosine-phosphatase inhibitor pervanadate (100 μM for 30 min). When Syk-GFP was co-transfected with tau, tau immunoprecipitates contained a ∼ 100-kDa tyrosine-phosphorylated protein, likely to contain Syk-GFP since this enzyme is tyrosine phosphorylated (Fig. 2A). This was confirmed by blotting with Syk antibody after membrane stripping (Fig. 2A). This co-immunoprecipitation of Syk with tau is indicative of Syk and its substrate being in the same protein complex. In order to exclude any effects of the V5 and GFP tags fused with tau and Syk, we wished to repeat this experiment using vectors expressing native proteins. The untagged and shortest isoform of tau (0N3R) was phosphorylated on tyrosine when co-transfected with native 72 kDa Syk (Fig. 2B), as compared with the transfection of tau alone. As in Fig. 2A, an additional 72-kDa band corresponding to phospho-Syk was visible on the phosphotyrosine blot when Syk was co-transfected with tau. The co-immunoprecipitation is therefore confirmed with native proteins. Taken together, these results demonstrate that Syk tyrosine kinase can both phosphorylate and co-immunoprecipitate with tau in cells, consistent with the direct phosphorylation of the substrate demonstrated in vitro (Fig. 1). The binding of Syk to both the shortest and longest isoform of tau suggests that it is independent of tau splicing.
Tau has been shown to be tyrosine phosphorylated on residue 18, 197 and 394 [6,8–10]. To map the tyrosine residue(s) phosphorylated by Syk in tau, Syk was co-transfected into CHO cells along with wild-type or mutant tau constructs in which individual tyrosines were replaced by phenylalanine (Y18F, Y197F and Y394F). These cDNA constructs have been described in detail previously [8]. The only such tyrosine mutation that resulted in a strong decrease in tau tyrosine phosphorylation was Y18F (Fig. 3). Tyrosine phosphorylation of the Y197F and Y394F constructs were not significantly different from the wild-type control (Fig. 3). This clearly demonstrates that Y18 is the major tyrosine phosphorylation site for Syk. In addition, the binding of Syk was independent of the tyrosine phosphorylation state of tau (Fig. 3), suggesting that the SH2 domains of Syk are not involved in its binding to tau.
As Fyn and Syk phosphorylate the same tyrosine residue on tau, we decided to perform a time course of phosphorylation of tau by the two kinases under conditions designed to favour the measurement of stoichiometry. After 30 min, Syk had incorporated an average of 0.23 mol of phosphate/mol of tau and Fyn had incorporated an average of 0.25 mol of phosphate/mol of tau. These results show that Syk and Fyn phosphorylate tau with the same efficacy (Fig. 4).
Our demonstration of binding of tau to Syk is consistent with tau being a specific substrate for Syk and for their involvement in a cell-signaling pathway in neurons. Interestingly, Syk plays an important role in signalling events of neurite induction and outgrowth in neuronal cell lines [16]. It is thus tempting to speculate that the phosphorylation of tau by Syk could be involved in neurite outgrowth. Our study also reinforces the role of Syk in neurodegenerative disorders. Remarkably, Y18 has been shown to be phosphorylated in NFT, suggesting that, like Fyn, Syk could be critical in the pathophysiology of AD [6,7,17]. Although early studies suggested a clear distinction between ‘tauopathies’ and ‘synucleinopathies’, more recent studies demonstrate that there is often an overlap in the pathological findings in these disorders [18–20]. Syk binds to and phosphorylates both tau and synuclein, suggesting that this kinase could be a link between synucleinopathies and tauopathies. In conclusion, our findings significantly contribute to the understanding of the signaling pathways involving tyrosine phosphorylation of tau in both physiological and pathological conditions. Further elucidation of the functional relationship between tau and the Syk in neurons could provide critical insights into the role of tau in cell signaling as well as the role of tau in neurodegenerative processes.

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Spanish health benefits for services of curative care

This contribution presents entitlements and benefits, decision criteria, and involved actors for services of curative care in Spain. It describes basic benefits included in the category of curative care defined by the central government and any additional benefits that some autonomous communities (ACs) have included to enlarge their own basket. It is concluded that there is no specific and explicit benefit catalogue. As no user charges exist for this category, waiting times serve as the main cost containment tool. There is a need for further legislation, as inequalities may increase across the territory as a matter of fact. Inequalities in access to health care resources between ACs are not due to differences in health baskets but mainly to the availability of technologies.
The principal actors responsible for defining of benefit catalogues from the services of curative care in Spain are the Central Government and the autonomous communities (ACs). Law 16/2003 [1] states that the Spanish health benefit basket should serve as a minimum for all Spanish residents to ensure the equality of access and benefits among them irrespective of their location. Nevertheless, as ACs may vary the allocation of their funding, some may decide to offer additional or new benefits even including those that are explicitly excluded on national level by Royal Decree 63/1995 [2] and Law 16/2003.
The central government’s main responsibilities are as follows: (a) general coordination and basic health legislation, (b) financing of the system and the regulation of the financial aspects of social security, (c) definition of a benefit basket guaranteed by the National Health System (NHS), (d) international health, (e) pharmaceutical policy, (f) health professionals undergraduate and postgraduate training, and (g) human resources policies regarding civil servants. A number of ministries (Economy and Finance, Public Administration, Labor and Social Affairs, Education and Culture, Environment, Justice, and Defense Ministries) share these responsibilities and therefore determine the health benefit basket, although the Ministry of Health and Consumer Affairs plays the main role in finally determining any health policies.
Regional governments (ACs) have the responsibility for planning, financing, and providing health care services (including an enlargement of the basic health benefit basket), social and community care, and public health. Another important agent in coordinating and evaluating new benefits is the Institute of Health Carlos III. Local governments are responsible for home-based nursery and social services. Coordination takes place in the Inter-Territorial Council of the NHS, and advisory committee comprising representatives from the central and regional governments that is responsible for ensuring the equality of benefits among and access across regions.
The present contribution focuses on services of curative care, excluding services such as rehabilitative care and goods dispensed to outpatients. The other categories have been studied in a detailed report as part of the Health Basket Project and are presented in Tables 1 and 2, including the benefit defining criteria. We first present an overview of the basic benefits that all ACs should provide and describe the additional benefits that some ACs include in their own basket. We then discuss the benefit catalogues and their actual implementation.Table 1 Benefit-defining laws/decrees and catalogues and the implicit regulation by sector. IGeneral Health ActRoyal Decree 63/95Law 16/03Ministerial Decree 30 April 1997Ministerial Decree 3 June 1998Ministerial Decree 3 March 1999Functional categories  HC.1.1+++–––  HC.1.2++–––  HC.1.3+++–––  HC.1.3.1+++–––  HC.1.3.2++–––  HC.1.3.3+++–––  HC.1.3.4+++––  HC.1.4+++–++  HC.2.1+++–––  HC.2.2+++–––  HC.2.3+++–––  HC.2.4––––––  HC.3–++–––  HC.4.1–++–––  HC.4.2–++–––  HC.4.3–++–––Legal statusLawPresidential DecreeLawMinistry DecreeMinistry DecreeMinistry DecreeDecision makerParliamentGovernmentParliamentGovernmentGovernmentGovernmentDegree of explicitnessa132333Positive/negative definition of benefitsPPPPPPOriginal purposeGeneral definition of entitlementsBasketEquality among citizens and ACsBasketBasketBasketCriteria used for defining benefits  Need++++++  Costs–+++++  Effectiveness–+++++  Cost-effectiveness––––––  Budget––––––UpdatingNoNoNoRegularlyRegularlyRegularlya 1, all necessary; 2, areas of care; 3, itemsTable 2 Benefit-defining laws/decrees and catalogues and the implicit regulation by sector. IIGeneral Health ActRoyal D 1663/1995Law 16/2003Decree 3157/1966Law 25/1990Royal Decree 83/1993Royal Decree 1663/1998Royal Decree 1348/2003Ministerial Decree 18 January 1996Functional categories  HC.5.1+––+++++–  HC.5.1.1––––+––––  HC.5.1.2––––+––––  HC.5.2–––––––––  HC.5.2.1–––––––––  HC.5.2.2–+––––––+  HC.5.2.3––––––––+  HC.5.2.4––––––––+  HC.6.1–++––––––  HC.6.2–+–––––––  HC.6.3+++––––––  HC.6.4+++––––––  HC.6.5–––––––––Legal statusLawPresidential DecreeLawLawLawPresidential DecreePresidential DecreePresidential DecreeMinisterial DecreeDecision makerParliamentGovernmentParliamentParliamentParliamentGovernmentGovernmentGovernmentGovernmentDegree of explicitnessa132123333Positive/negative definition of benefitsPPPPPNNNPOriginal purposeGeneral definition of entitlementBasketEquality among citizens and ACsGeneral definition of entitlementTarget ruleExclusionExclusionExclusionBasketCriteria used for defining benefits  Need+++++–––+  Costs–++–+++++  Effectiveness–++–+++++  Cost-effectiveness–––––––––  Budget–––––––––UpdatingNoNoNoNoNoRegularyRegularyRegularyRegularya 1, all necessary; 2, areas of care; 3, items
Definition and structure of the main benefit basket for curative care
With regards to benefit coverage the Spanish Constitutional Act stipulates Spanish citizenship as the only precondition for access to the range of health care benefits. Parliamentary ratification on 18 December 1997 [3] extended coverage to the entire resident population. The major differences between ACs are related to foreigners.
The fundamental act on health benefit regulation is Royal Decree 63/1995 (20 January) on the organization of health services provided by the NHS (Fig. 1). It provides inclusion and exclusion criteria and defines the current level of provision as the guaranteed basic health care basket in a comprehensive form. It contains a checklist of services that constitute the entitlement, establishes certain exclusion criteria, and limits the provision of certain services due to the limited resources available to the NHS. It also stipulates conditions for the introduction of new technologies and procedures as well as new entitlements. Finally, it prescribes coordination between health and social care.Fig. 1 Royal Decree 63/1995
Services, activities, and procedures are excluded under any of the following circumstances: (a) There is insufficient scientific evidence of their clinical safety and efficacy or if they are clearly outdated. (b) They have not sufficiently proven their effective contribution to prevention, treatment, or cure illnesses, preservation or improvement of life expectancy, self-help, or elimination or reduction of pain and suffering. (c) They are constitute merely activities such as those of leisure, rest, comfort, sport, cosmetic improvement, or spa.
The Spanish NHS has not yet in fact defined a basic basket of services, although Royal Decree 63/1995 established a general framework of benefits, as did the Law 16/2003 of 28 May (Fig. 2). This law on “Cohesion and Quality of the NHS” is a reform that built on the previous act with the purpose of coordinating a strongly decentralized activity. Its aim is to establish the legal framework for the coordination and cooperation of the Public Health Administrations guaranteeing equality, quality, and participation in the system. It also introduced the establishment of a general procedure for revisions on the content of the health benefit basket. Since 2002 all ACs have health service responsibilities. They manage their regional health care system and also have related legislative responsibilities. These regional health care baskets must include at least the benefits defined by the NHS. In this sense Law 16/2003 is intended to sustain the coordination and the basic guarantees between all the territories.Fig. 2 Law 16/2003
Chapter I of Law 16/2003 refers to health care benefits of the NHS and defines a catalogue that incorporates the benefits defined by Royal Decree 63/1995, public health benefits. The catalogue of benefits is defined as a set of preventive, diagnostic, therapeutic, rehabilitative services, and direct health promotion to citizens, which includes public health, primary and specialized care, social and community care, dietetic urgencies, pharmacy, orthoprosthesis, products, and sanitary transport benefits. It guarantees the right of all citizens to obtain a second medical opinion, the right to receive medical assistance in one’s own AC of residence within a maximum time along, and the right to receive the defined benefits of the NHS under the same conditions and guarantees as residents in other ACs.
As a further development of the 16/2003 Law the Spanish Ministry of Health is currently drafting a Royal Decree to establish the NHS health benefit basket and the means for updating it. The purpose of the Royal Decree is to guarantee equality and appropriate care by the NHS. The ACs approve their own baskets by always including all the services of the NHS health benefit basket since these services are considered a basic need and common to all users of the NHS. They can enlarge their own baskets with additional technical services, technologies, and procedures. New benefits added to the basic basket are evaluated by the Ministry of Health through the Evaluation Agency of Health Technologies (“Agencia de Evaluación de Tecnologías Médicas”) and the Carlos III Health Institute in collaboration with other evaluation agencies proposed by the ACs and the Inter-Territorial Council of the NHS.
Contents of the benefit baskets for curative care
Inpatient curative care
The above regulation affecting inpatient curative care defines the benefits covered by the NHS which ought to be provided by all ACs (free of any user payment) such as: specialized health care in hospitals, which includes medical attention, surgery, obstetric, and pediatric services for acute conditions, worsening of chronic conditions or delivery of recommended diagnostic treatments or procedures; organs, tissues and cells of human origin transplants according to the special legislation as long as there is the evidence for therapeutic effectiveness; palliative care to terminally ill patients; and mental health care and psychiatric assistance, which includes clinical diagnosis and follow-up and the prescribed group of family psychotherapy if it implies hospitalization when required. Hospitalizations of short or medium periods of time take place in the psychiatric departments of general hospitals. Inpatient mental long-term care services take place at psychiatric hospitals, which are part of the long-term care network (local authorities).
The main benefit exclusions are mental inpatient care (included only upon referral) and transplants of hair, nails, and placenta. Additional benefits on the part of ACs include sex/gender change surgery intervention, such as that now offered in Andalusia. Andalusia also offers epidural anesthetics during birth. The other main benefit extension is related to euthanasia. Catalonia, Galicia, and Extremadura have regulated and included the vital testament as a benefit.
Day cases of curative care
The entitlements concerning day cases of curative care define the service groups included in the Spanish benefits basket as: specialized ambulatory health care consultations that could include minor surgical procedures; specialized ambulatory health care delivered in “day hospitals” for patients in need of continuous specialized care, physician’s or nursing services, including major surgery as long as no hospitalization is required; and hemotherapy. The following services are explicitly excluded: plastic surgery when it is not related to accident, disease, or congenital malformation; sex change surgery, except for those cases in which it is necessary to repair pathological intersexual conditions.
Services of outpatient care
Outpatient care is generally provided in primary care centers or specialist care centers (outpatient clinics). There is a wide disparity in the number and use of the latter among ACs. These categories of care are regulated both at national and AC levels. The outpatient benefit basket includes the following service groups (all free of user charges): health assistance for health consultation services and centers including basic medical and diagnostic services (curative) primary care; health services provided at home; indication or prescription and the compliance, in relevant cases, with examinations and basic diagnostic measures as indicated by the primary care physician; the administration of parenteral treatments, cures and minor surgery; initial mental assistance is provided in mental health centers, where patients are being evaluated, assisted, or referred to other assistance services. Benefits provided include: pharmacological and psychotherapy treatments, crisis interventions, etc. Mental emergencies can also be treated at the hospital emergency service; emergency primary health care which will be provided to persons of any age and delivered continuously 24 h a day, through medical and nursing services, on an outpatient basis or at the domicile of the patient if the situation requires to do so; primary dental health care, including dental health and hygiene information and education, preventive and assistance measures; application of topical fluoride, obstructions, sealing of fissures or other services for the infant population according to the annual financial budget and special programs for dental health, treatment of acute orthodontic problems including dental extractions, and preventive exploration of the oral cavity in pregnant women; renal lithotripsy, interventionist radiology, and radiotherapy. Certain services such as complex diet therapy are not free of charge but are also not explicitly excluded from the Spanish health care benefit basket. Other services such as spa treatments, rest cures, psychoanalysis, and hypnosis are explicitly excluded.
In this category of care many ACs offer additional services as part of their benefit catalogues. At primary care level the Basque Country, Extremadura, Castilla La Mancha, and Catalonia have passed several decrees; at specialized outpatient care level Navarre offers obstetrics, gynecological and family planning such as menopause care; Valencia provides reactive strips for determining glucose in blood and urine to diabetics; and at special treatment level the Balearic Islands offer neurological reflexology in cooperation with the Kovacs Foundation. It must also be noted that Catalonia has started a pilot experience in offering acupuncture for arthritis treatment.
Nine ACs also provide free dental care for some groups: Navarre, Basque Country, Andalusia, Extremadura, Castilla La Mancha, Cantabria, Castilla León, Murcia, and Galicia. Catalonia offers periodic rinsing of a fluorine solutions program for primary school students and those taking the first two courses of secondary school. As all these catalogues are quite similar, we present below the dental care benefit catalogue of Castilla La Mancha (Fig. 3). The Infant-Young Dental Care Plan defines the group of measures and activities, both preventive and welfare, as well as any procedures to provide dental care. Benefits are classified into: oral check-up, basic dental treatments, emergency dental treatments, special dental treatments, and orthodontics.Fig. 3 Dental care catalogues: the example of Castilla La Mancha
Services of curative home care
As part of the entitlements of services of curative home care, oxygen therapy service at home is provided at authorized and specialized centers capable of carrying out gasometry and spirometry. The Ministerial Decree of 3 March 1999 [4] elaborates these services, considering the following benefits: (a) oxygen therapy at home, (b) mechanical ventilation at home, (c) ventilation treatment of the sleep apnea symptoms, and (d) aerosol therapy. Enteral nutrition is also provided at home. This service is made available to patients who suffer from the following symptoms: swallowing, transit, digestion or absorption of foods in its natural form problems or when special requirements of energy and/or nutrients exist that cannot be covered with foods of daily consumption.
Royal Decree 63/1995 has been developed with regard to benefits with diet products in two ways: regulating complex diet therapy and regulating enteral nutrition at home. Complex diet therapy is dealt with in the Ministerial Decree of 30 April 1997 [5] which specifies the relationship of congenital metabolic upheavals of carbon hydrates and amino acids included in this benefit and the type of diet in each case. It also prescribes these treatments to be performed by a special physician in hospital units, specifically authorized by the appropriate Health Administration. Enteral nutrition at home is regulated by the Ministerial Decree of 2 June 1998 [6], which defines the requirements and the patient’s clinical situation for this. There are also regulations at the national and interterritorial council levels as well as in six ACs that have developed their own dietetic regulations.
The Ministerial Decree of 3 March 1999 was passed upon the proposal of a panel of experts (Fig. 4). This elaborates Royal Decree 63/1995 regarding the techniques of oxygen therapy at home financed by the NHS and clarifies and standardizes the criteria, clinical situations, and other matters for the direction, prescription, and provision of these benefits. It defines the oxygen therapy techniques that can be carried out at home in terms of their financing by the NHS and establishes the basic requirements that justify their prescription. The Ministerial Decree of 3 March 1999 thus provides a common basis upon which the health service providers must develop their own procedure for providing and supervising these benefits. The Decree actually does not provide a benefit catalogue as such because the individual physician determines the specific treatment in each case. In addition there are also specific regional oxygen therapy regulations in four ACs (Andalusia, Canary Islands, Navarre and Valencia).Fig. 4 Oxygen therapy at home
Services of MUFACE catalogue
All civil servants and beneficiaries are entitled to receive benefits as determined by the MUFACE catalogue. This benefit basket is virtually equivalent to that of the NHS but include some additional benefits. The principal difference is related to copayments for pharmaceuticals. In addition to particular features for medical assistance abroad, orthoprosthetic, dental care, ocular, pharmaceutical, and sociosanitary benefits, services such as vaccinations are included free of charge. Specialized health care includes all the medical and surgical specialties, both in outpatient and inpatient sectors. The specialties are structured in four levels according to their complexity and geographic scope, sanitary transport, oxygen therapy, and aerosol therapy at home.
Discussion
The way in which the Spanish benefit catalogue is defined is characteristic of tax-financed NHS health care systems. A system such as that in Spain which guarantees universal coverage is not completely developed until it is specifies a minimum basket of health care services. However, as no specific and explicit benefit catalogue is being developed, there remains a need for further legislation. The Ministry of Health and Consumption is moving towards an explicit process of decision making in health benefit planning instead of defining a detailed benefit catalogue.
As is the case in many countries, the possibility of using regional plans as the basis for resource allocation and capacity planning has not yet been considered. An additional problem in Spain is the fact that the majority of health care plans offer unrealistically long lists of objectives which are often not sufficiently based on available epidemiological and cost-effectiveness evidence, and their efficacy is seldom evaluated. They cannot be considered as benefit catalogues since they are not sufficiently strong legal instruments, and therefore users are not able to claim the objectives contained in the health plans as benefit rights.
Then purpose of the Spanish health benefits basket is twofold, as it was initially established to serve more as a budgetary than as a cost containment measure when created by the Social Security act [7]. However, on the other hand, as Royal Decree 63/1995 and the 2003 Cohesion Law recognize, they are merely an ordering of benefits as they try only to describe what is being offered in practice.
Two major aspects of the Spanish health care system are: (a) A more explicit definition of the benefit basket is needed as the present vague definition leaves the final decisions to practitioners, making expenditure very volatile, and having as the sole restriction that of waiting times. (b) Greater transparency is needed in the process of approval/rejection of new benefits, towards agents in the health sector, and towards citizenship along with the need to include economic considerations in the decision process. In Spain it still remains the case: “all for everyone and almost everything for free.” This obviously leads to long waiting times and thus a dual system (public-private) for those able to skip the long queues and high public pressure over resources devoted to the NHS. Furthermore, since there are no user payments, waiting lists serve as the mechanisms restricting demand for health services. For the first time, the Catalan Health Service is now studying the possibility of introducing a copayment system in primary care (fixed payment per visit).
There are still some remaining problems in the Spanish benefit basket. In terms of equality, the fact that MUFACE-type schemes have not been fully integrated to the NHS provides additional benefits for these groups. There is a lack of real control over the managers of the health care services. Autonomy of decisions by practitioners may mean an obstacle to the early evaluation of new technologies since under uso tutelado (monitored use) it is physicians who start using them. In the present financing system there is no relationship between this and the benefits provided, nor a control mechanism over what is provided in reality.
Moreover, the information systems are clearly the main problem in the decentralization process. To overcome this problem the Ministry of Health has recently introduced an institution for monitoring the health system. Even the appearance of new benefit differences across ACs will not affect equality such as it may affect clinical practices and access to equipment. Therefore the main purposes of the Spanish health policy are to promote common clinical guidelines and evaluation processes with a single registry on new infrastructure and the creation and regulation of a common procedure for new benefit approvals.
In the future the central government is not expected to increase benefits but rather the quality of the existing ones, through new technologies and clinical procedures. Improvements are expected regarding the provision of services (e.g., waiting times), better user information, and increased range of choices. Probably the most important task is the development of a long-term care or dependency system and a real network of mental health.
Future reforms taken by the different ACs will likely address the benefits basket. Dental care and natural and alternative medicines are probably the first services that will be addressed. The definition of a basic long-term social and community care benefit basket similar to that in health care will also be a priority for the central government. Nevertheless, as we can see in Table 3, new benefits have very little impact on the health expenditure of ACs.Table 3 Spending in the provision of health care services not included in the benefit catalogue of the National Health System (Royal Decree 63/1995): proportion of total health care services spending19992000200120022003Total0.150.130.130.120.11Andalusia0.400.320.320.300.29Aragon–––––Asturias–––0.00030.0007Balearic Islands––––0.02Canary Island–––––Cantabria–––––Castilla y León–––––Castilla–La Mancha–––––Catalonia0.400.380.360.320.27Valencia–––––Extremadura––––0.04Galicia–––––Madrid–––––Murcia––––0.05Navarre0.470.440.470.460.49Basque Country0.200.180.190.180.19La Rioja–––––Ceuta–––––Melilla–––––
Finally, in Spain, the growth of public health expenditure is due to both the increase in input prices and the received average benefits per capita. The growth of public health care expenditure is largely attributable to the benefits provided. The yearly change in health care benefits was responsible for 1.86% of the 6.73% annual average increase in health expenditure between 1991 and 2003.

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