The effect of long-term hypogonadism on body composition and morphometry of aged male Wistar rats.

Clinical studies show that hypogonadism in the aging male is associated with obesity and osteoporosis. Experimental studies are mostly conducted on relatively young adult animals and the induced hypogonadism lasts for a relatively short time. The present study aimed to describe the effect of long-term hypogonadism beginning in puberty on body composition, morphometry, and bone mineral density in aged male rats. Morphometric measurements and dual-energy X-ray absorptiometry were conducted at the age of 30 months on control and gonadectomized males. Long-term hypogonadism did not affect body weight, but led to a higher fat mass (by 26 %), lower lean mass (by 44 %), shorter body length (by 9 %), and anogenital distance (by 26 %), as well as to lower tail circumference (by 15 %) in comparison to control males. Lower bone mineral density (by 13 %) and bone mineral content (by 15 %) were observed in gonadectomized males. Results showing sarcopenic obesity and osteoporosis in this model of long-term hypogonadism might mimic the situation in aging males better than the widely used short-term hypogonadism induced in young animals. The morphometric analysis could potentially be a useful tool to study normal weight obesity without the need for specific equipment.

Metabolic and renal effects of dietary advanced glycation end products in pregnant rats - a pilot study.

Thermally processed food contains advanced glycation end products (AGEs) including N(epsilon)-(carboxymethyl)lysine (CML). Higher AGEs or circulating CML were shown to be associated with pregnancy complications such as preeclampsia and gestational diabetes. It is unclear whether this association is causal. The aim of our study was to analyze the effects of dietary CML and CML-containing thermally processed food on metabolism in pregnant rats. Animals were fed with standard or with AGE-rich diet from gestation day 1. Third group received standard diet and CML via gavage. On gestation day 18, blood pressure was measured, urine and blood were collected and the oral glucose tolerance test was performed. Plasma AGEs were slightly higher in pregnant rats fed with the AGE-rich diet (p=0.09). A non-significant trend towards higher CML in plasma was found in the CML group (p=0.06). No significant differences between groups were revealed in glucose metabolism or markers of renal functions like proteinuria and creatinine clearance. In conclusion, this study does not support the hypothesis that dietary AGEs such as CML might induce harmful metabolic changes or contribute to the pathogenesis of pregnancy complications. The short duration of the rodent gestation warrants further studies analyzing long-term effects of AGEs/CML in preconception nutrition.

Markers of oxidative stress and antioxidant status in the plasma, urine and saliva of healthy mice.

Oxidative stress markers are usually measured in plasma, a stable environment for biomarkers. Blood collection is invasive, but the use of alternative biofluids is limited, due to high variability. In this study, we aimed to establish reference values for oxidative stress markers in plasma, urine and saliva of adult, healthy mice and to identify some sources of variability. Samples were obtained from 41 female and 37 male adult, healthy mice of the CD-1 strain, aged 95-480 days, weighing 21-55 grams. Reference ranges of TBARS (thiobarbituric acid reactive substances), AOPP (advanced oxidation protein products), fructosamine, GSH/GSSG (reduced and oxidized glutathione) ratio, TAC (total antioxidant capacity), and FRAP (ferric reducing antioxidant power) were measured in plasma and urine, and TBARS, GSH/GSSG ratio, TAC and FRAP in saliva, using standard spectrophotometric and fluorometric methods. Salivary GSH/GSSG and urinary AOPP were higher in females. Urinary fructosamine, GSH/GSSG and FRAP were higher in males. Urinary TAC and FRAP negatively correlated with age, and urinary GSH/GSSG positively correlated with weight. We determined that urine and saliva can be obtained non-invasively from mice, in sufficient amounts for reliable oxidative status assessment. Further studies are needed to uncover whether these biofluids reflect systemic oxidative status in diseases.

Plasma markers of oxidative status were associated with increasing continuous cardiometabolic risk scores in healthy students aged 16-20 years without central obesity.

AIM: We studied the association between increased cardiometabolic risk and markers of oxidative status and glycation in apparently healthy subjects who did not present with central obesity. METHODS: From 2011 to 2012, we recruited 2064 students (53% girls) aged 16-20 years from Western Slovakia. Their continuous metabolic syndrome scores (MSS) were calculated as a mean of the sum of the z-scores of waist-to-height ratio, mean arterial pressure, triacylglycerols, high-density lipoprotein-cholesterol and quantitative insulin sensitivity check index. Plasma markers of protein glycation and oxidation, lipid peroxidation and total antioxidant status were analysed. RESULTS: In both genders, advanced oxidation protein products (AOPPs) increased across the MSS quintiles (p < 0.001). AOPPs and fructosamines were significant predictors of the MSS in both genders. Moreover, high-sensitivity C-reactive protein, leukocyte counts and advanced glycation end-products (AGEs) contributed significantly in girls. Triacylglycerols, fructosamines, AGEs and total antioxidant capacity correlated significantly with AOPPs in both genders. CONCLUSION: Advanced oxidation protein products may act as inflammatory mediators that contribute to the development of cardiometabolic afflictions. Determining these may provide information related to cardiometabolic risk and represent potential target to reduce or prevent irreversible oxidative stress-induced cellular damage.

Continuous metabolic syndrome score (siMS) enables quantification of severity of cardiometabolic affliction in individuals not presenting with metabolic syndrome.

Assessment of degree of cardiometabolic affliction in subjects not presenting with metabolic syndrome (MS) yet, would be helpful in the management of preventive health maintenance. OBJECTIVES: To evaluate continuous metabolic syndrome score (siMS) in estimation of severity of cardiometabolic affliction in individuals not presenting with MS. METHODS: We analyzed data from 3166 volunteers (56 % females) aged ≥ 16 years. siMS score was calculated as waist/height/0.5 + fasting plasma glucose (FPG)/5.6 + triacylglycerols (TAG)/1.7 + systolic blood pressure (SBP)/130 - high-density lipoprotein cholesterol (HDL-C)/1.02 (males) or 1.28 (females). In siMS quintiles, numbers of individuals presenting with 0-to-5 MS components were calculated. MS was considered as the presence of any 3 out of its 5 components. RESULTS: 33 % of participants without MS scored ≥ 4th quintile; 13 % of those free from MS components; 49 % of participants presenting with 1, and 83 % of those displaying 2 MS components. 11 % of individuals presented with MS, all but 1 displayed siMS within the 2 upper quintiles. CONCLUSIONS: Considerable proportion of individuals without MS presented with siMS in range displayed by individuals presenting with MS. SiMS might be useful in estimation of severity of cardiometabolic affliction prior to manifestation of MS, to identify individuals requiring early intervention to counteract developing pathological processes (Tab. 1, Ref. 21).

Vitamin D status in apparently healthy medication-free Slovaks: Association to blood pressure, body mass index, self-reported smoking status and physical activity.

BACKGROUND: Vitamin D plays a role in protecting against chronic degenerative diseases. Slovak adults present one of the highest cardiovascular mortality rates among 27 EU countries. OBJECTIVES: We asked whether the 25(OH)D3 status in apparently healthy medication-free Slovaks deteriorates upon ageing, and in the presence of cardiometabolic risk factors. METHODS: We studied the impact of blood pressure, overweight/obesity, smoking, and physical activity on 25(OH)D3 levels determined using RIA method in 578 (5-81 years old) subjects. RESULTS: The average level of 25(OH)D3 was 36±17 ng/ml. A proportion of 15 % of participants were 25(OH)D3­deficient (≤20 ng/ml), 26 % presented insufficient (20-to-30 ng/ml), and 59 % satisfactory (> 30 ng/ml) levels. Neither mean 25(OH)D3 levels, nor the prevalence of hypovitaminosis D showed age dependence. Physically active normotensive non-smokers presented the highest (41±19 ng/ml), and their smoking counterparts with elevated BP the lowest 25(OH)D3 levels (30±12 ng/ml). CONCLUSION: In apparently healthy medication-free Slovaks the prevalence of hypovitaminosis D is high. Vitamin D status does not deteriorate in course of healthy ageing. Physical activity, normotension, and non-smoking status are associated with favorable vitamin D status while low 25(OH)D3 levels are associated with multiple cardiometabolic risk factors. Further studies in subjects at high cardiovascular risk are needed to elucidate the potential association of hypovitaminosis D with high cardiovascular mortality in Slovak adults (Tab. 1, Fig. 4, Ref. 42).

Is vitamin D deficiency related to accumulation of advanced glycation end products, markers of inflammation, and oxidative stress in diabetic subjects?

OBJECTIVES: In diabetes accumulated advanced glycation end products (AGEs) are involved in the striking cardiovascular morbidity/mortality. We asked whether a hypovitaminosis D associates with an increased formation and toxicity of AGEs in diabetes. METHODS: In 276 diabetics (160 M/116 F, age: 65.0 ± 13.4; 43 type 1,T1DM, and 233 type 2 patients, T2DM) and 121 nondiabetic controls (60 M/61 F; age: 58.6 ± 15.5 years) routine biochemistry, levels of 25-hydroxyvitamin D3 (25-(OH)D), skin autofluorescence (SAF), plasma AGE-associated fluorescence (AGE-FL), N (ε) -(carboxymethyl)lysine (CML), soluble receptor for AGEs (sRAGE), soluble vascular adhesion protein-1 (sVAP-1), high sensitive C-reactive protein (hs-CRP), and renal function (eGFR) were determined. RESULTS: In the diabetics SAF and AGE-Fl were higher than those of the controls and correlated with age, duration of diabetes, and degree of renal impairment. In T2DM patients but not in T1DM the age-dependent rise of SAF directly correlated with hs-CRP and sVAP-1. 25-(OH)D levels in diabetics and nondiabetics were lowered to a similar degree averaging 22.5 ng/mL. No relationship between 25-(OH)D and studied markers except for sVAP-1 was observed in the diabetics. CONCLUSION: In diabetics hypovitaminosis D does not augment accumulation of AGEs and studied markers of microinflammation and oxidative stress except for sVAP-1.

Towards an alternative testing strategy for nanomaterials used in nanomedicine: lessons from NanoTEST.

In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it still remains unclear how exactly NPs interact with their cellular targets. Size, surface, mass, geometry, and composition may all play a beneficial role as well as causing toxicity. Concerns of scientists, politicians and the public about potential health hazards associated with NPs need to be answered. With the variety of exposure routes available, there is potential for NPs to reach every organ in the body but we know little about the impact this might have. The main objective of the FP7 NanoTEST project ( www.nanotest-fp7.eu ) was a better understanding of mechanisms of interactions of NPs employed in nanomedicine with cells, tissues and organs and to address critical issues relating to toxicity testing especially with respect to alternatives to tests on animals. Here we describe an approach towards alternative testing strategies for hazard and risk assessment of nanomaterials, highlighting the adaptation of standard methods demanded by the special physicochemical features of nanomaterials and bioavailability studies. The work has assessed a broad range of toxicity tests, cell models and NP types and concentrations taking into account the inherent impact of NP properties and the effects of changes in experimental conditions using well-characterized NPs. The results of the studies have been used to generate recommendations for a suitable and robust testing strategy which can be applied to new medical NPs as they are developed.

Reference values of skin autofluorescence as an estimation of tissue accumulation of advanced glycation end products in a general Slovak population.

AIMS: For decades, Slovakia has maintained a prominent place in mortality rates from cardiovascular diseases among European Union (EU-27) countries. Determination of skin autofluorescence serves as an estimate of tissue accumulation of advanced glycation end products--substances accumulating in tissues and body fluids that play a pathophysiological role in age-related diseases and their complications, such as diabetes. METHODS: In 1385 apparently healthy Slovakian subjects aged from a few days old to 77 years, skin autofluorescence was determined using an advanced glycation end product reader and compared with reference data from Dutch Caucasians. The impact of the weekly frequency of recreational physical exercise on skin autofluorescence was investigated in the adults, and the impact of feeding regimen in the infants. RESULTS: With the exception of 10- to 19-year-olds, Slovaks had lower skin autofluorescence values in comparison with the Dutch Caucasians. In healthy non-smokers, physical exercise for > 30 min/day performed ≥ 3 times/week was associated with lower skin autofluorescence levels. In infants, breastfeeding (advanced glycation end product-poor diet) was associated with lower skin autofluorescence levels in comparison with consumption of infant formulas (advanced glycation end product-rich diet). CONCLUSIONS: Reference ranges of skin autofluorescence in Slovak Caucasians, detailed for paediatric age groups, are provided. Our data show that, in healthy adults, regular physical exercise associates with lower skin autofluorescence. Infants fed or weaned from infant formulas (advanced glycation end product-rich diet) have higher skin autofluorescence than their breast milk-consuming counterparts. It is unclear why Slovaks have lower skin autofluorescence compared with a Dutch population with lower cardiovascular mortality rates. Reference data on skin autofluorescence from diverse populations are needed for the precise clinical interpretation of skin autofluorescence measurements.

[An association between microalbuminuria and obesity in healthy adolescents - preliminary results from the "Respect for Health" study]. / Vztah mikroalbuminúrie a obezity u zdravých mladistvých - predbezné výsledky stúdie "Respekt pre zdravie"

BACKGROUND: Microalbuminuria is a marker of present/future cardiovascular and/or renal disease. The roots of these diseases extend back into childhood. Data on renal excretion of albumin (albumin/creatinine ratio - ACR) and the prevalence of microalbuminuria in apparently healthy adolescents are scares. METHODS AND RESULTS: We determined ACR and the prevalence of microalbuminuria in 15-to-19-years-old adolescents (n = 846, 482 boys), in association with markers of obesity. ACR (0.43; 0.29-0.67 mg albumin/mmol creatinine vs 0.35; 0.25-0.51 mg albumin/mmol creatinine, p < 0.001), and the prevalence of microalbuminuria (3.6 % vs 1.2 %, χ2: p = 0.024) were higher in girls than in boys. In underweight subjects, particularly boys, ACR was significantly higher if compared with the overweight//obese subjects. ACR correlated inversely with the markers of peripheral and central obesity. CONCLUSIONS: Prevalence of microalbuminuria in general population of adolescents was relatively low. Paradoxically, in the boys ACR showed an inverse relationship to markers of nutritional status. Our data suggest the need of specific interpretation of data on ACR in the adolescents, and the need of further analysis of this (in the adults risk) marker in population of adolescents with regard to other important determinants of ACR, such as actual blood pressure, insulin sensitivity, etc.

Association of biochemical parameters and RAGE gene polymorphisms in healthy infants and their mothers.

BACKGROUND: The receptor for advanced glycation end-products (RAGEs) and its gene polymorphisms are implicated in the pathogenesis of different chronic diseases including diabetes and its complications. Infant formulas contain high amounts of advanced glycation end-products (AGEs) - the ligands of RAGE. METHODS: In this cross-sectional study, we examined the impact of G82S and -374 A/T polymorphisms in the gene encoding RAGE on standard blood chemistry, soluble (s)RAGE and inflammatory markers in 244 healthy infants (3-16months of age) and in 119 healthy mothers. Children were subdivided according to age (younger and older than 8months) and for the -374 A/T polymorphism according to the feeding regimen (breast-fed vs. infant formula-fed). RESULTS: Minor allele of the RAGE gene polymorphism G82S was associated with reduced plasma sRAGE in all age groups and with increased sICAM-1 in older children and mothers. Minor allele carrying mothers had decreased insulin sensitivity and HDL. The A allele of the RAGE gene promoter polymorphism -374 A/T was associated with higher indices of insulin resistance in young infant formula-fed, but not breast-fed children. In older, formerly infant formula-fed children signs of insulin resistance diminished, while formerly breast-fed children with A allele were more insulin sensitive. CONCLUSIONS: The phenotype of minor allele carriers in G82S is associated with reduced levels of protective sRAGE in healthy infants. With increasing age sICAM-1 levels increased and insulin resistance developed. In early childhood the phenotype of the -374 A/T polymorphism was diet-dependently associated with changes in glucose metabolism, which diminished with increasing age.

Neuronal activation in the CNS during different forms of acute renal failure in rats.

The effect of experimentally induced acute renal failure (ARF) on neuronal cell activation was investigated by immunohistochemistry for Fos and Fra-2 in the rat brain. ARF in rats was induced by bilateral nephrectomy (BNX), bilateral ureter ligature (BUL) and uranyl acetate injection with proper controls (sham-operation or saline injections, respectively). To follow the effect of the development of ARF, rats were killed 30 and 60 min, and 3, 12, 24 and 72 h after surgery, or 3 h to 12 days after uranyl acetate injections. In the BUL and BNX rats, urea and creatinine rose markedly in the plasma within 72 h, while in the uranyl acetate-injected rats the highest levels were observed on the 7th day, followed by a marked decline. At each time-point of the three different, experimentally induced ARF, the presence of Fos- and/or Fra-2-immunoreactive neurons was determined in 120 different brain areas and nuclei. In general, the 73 of 120 brain areas that showed time and intensity dependent activation in response to ARF can be classified into four groups: 1) biogenic amine (noradrenaline, adrenaline, histamine and 5-HT) expressing cell groups in the lower brainstem, 2) "stress-sensitive" forebrain areas, with regard to certain hypothalamic, limbic and cortical areas, 3) neuronal cell groups that participate in the central regulation of body and brain water and electrolyte homeostasis, including the circumventricular organs, and 4) central autonomic cell groups, especially visceral sensory cell groups in the brain, which are in primary, secondary or tertiary connections with renal afferents. Data presented here indicate that a wide variety of neurons in several regulatory mechanisms is affected by ARF-induced peripheral and central alterations.

Accumulation of free adduct glycation, oxidation, and nitration products follows acute loss of renal function.

Glycation, oxidation, and nitration of endogenous proteins occur spontaneously and these modifications are also present in foods. Increased levels of these chemical changes are associated with chronic renal failure; however, little is known about acute kidney failure. We measured these modifications of plasma protein and related free adducts in plasma following bilateral nephrectomy and bilateral ureteral obstruction. Advanced glycation end-product (AGE) residues of plasma protein were increased 3 h post-surgery, and thereafter slowly decreased in all groups, reflecting changes in plasma protein synthesis and transcapillary flow post-surgery. Ureteral ligation increased oxidation and nitration adduct residues. There were, however, marked increases in AGE, dityrosine, or 3-nitrotyrosine free adducts in both nephrectomized and ureter-ligated rats compared to rats that had undergone sham operations. There were lower modified adduct concentrations in the ureter-ligated compared to the nephrectomized rats, reflecting residual glomerular filtration and tubular removal. There was no increase in glycated, oxidized, and nitrated proteins. Glyoxal and methylglyoxal were also increased in both renal failure models. Our study shows that the acute loss of renal function and urinary excretion leads to the accumulation of AGE, oxidation, and nitration free adducts in the plasma.

Relation between different treatment modalities and genomic damage of end-stage renal failure patients.

BACKGROUND: Patients with end-stage renal disease display enhanced genomic damage. We investigated the relation between genomic damage and different treatment modalities. METHODS: In a longitudinal study two groups of patients were analyzed in monthly intervals. We assessed the initiation of hemodialysis in 5 conservatively treated patients, and a switch from hemodialysis to hemodiafiltration in 7 patients. DNA damage was investigated in peripheral blood lymphocytes by micronucleus frequency and by comet assay analysis. With regard to potential genotoxicity of advanced glycation end products (AGEs), levels of imidazolone A and AGE-associated fluorescence (AGE-FL) were determined. RESULTS: The initiation of hemodialysis did not alter the genomic damage. In patients who switched from hemodialysis to hemodiafiltration, a small but significant reduction in the comet assay but not in the micronucleus frequency was observed. Elevated plasma levels of imidazolone A and AGE-FL were not influenced by the treatment modalities. CONCLUSION: In our small patient group no major reduction of the elevated genomic damage could be reached. Disease factors not influenced by altered dialysis modalities may have contributed considerably in our patient group. The persisting high levels of DNA damage suggest a need for further improvement. Inhibiting AGE formation may be one promising way for the future.

Rooibos tea (Aspalathus linearis) partially prevents oxidative stress in streptozotocin-induced diabetic rats.

The aim of this study was to investigate the effects of rooibos tea as a natural source of a wide scale of antioxidants on the prevention and treatment of oxidative stress in streptozotocin-induced diabetic rats. Expected significant changes of biochemical parameters characteristic for experimental diabetic state were found in plasma and tissues eight weeks after single dose streptozotocin application. Administration of aqueous and alkaline extracts of rooibos tea (or N-acetyl-L-cysteine for comparison) to diabetic rats did not affect markers of the diabetic status (glucose, glycated hemoglobin and fructosamine). Besides the parameters characterizing hepatotoxic effect of streptozotocin, rooibos tea significantly lowered advanced glycation end-products (AGEs) and malondialdehyde (MDA) in the plasma and in different tissues of diabetic rats, particularly MDA concentration in the lens. From these results we can conclude that antioxidant compounds in rooibos tea partially prevent oxidative stress and they are effective in both hydrophobic and hydrophilic biological systems. Therefore, rooibos tea as a commonly used beverage can be recommended as an excellent adjuvant support for the prevention and therapy of diabetic vascular complications, particularly for protecting ocular membrane systems against their peroxidation by reactive oxygen species.

Paradox of circulating advanced glycation end product concentrations in patients with congestive heart failure and after heart transplantation.

OBJECTIVES: To analyse circulating concentrations of advanced glycation end products (AGEs) in patients with severe congestive heart failure (CHF) and after heart transplantation; to identify the potential contribution of kidney function to plasma AGE concentrations; and to determine whether AGE concentrations and parameters of oxidative stress are interrelated. METHODS AND RESULTS: Circulating N(epsilon)-(carboxymethyl)lysine (CML) and AGE associated fluorescence (AGE-Fl), lipid peroxidation, and glomerular filtration rate (GFR) were measured in a cross sectional study of 22 patients with advanced CHF, 30 heart transplant recipients, and 20 healthy controls. Compared with the controls, the CHF patients had decreased CML (mean (SEM) 467.8 (20.0) ng/ml v 369.3 (22.3) ng/ml, p < 0.01), AGE-Fl (mean (SEM) 302.2 (13.3) arbitrary units v 204.9 (15.7) arbitrary units, p < 0.01), and GFR (p < 0.01). CML was positively related to decreased total protein and serum albumin and negatively to body mass index (p < 0.01). In contrast, in the heart transplant group, impaired GFR was associated with a notable rise of both CML (mean (SEM) 876.1 (53.1) ng/ml, p < 0.01) and AGE-Fl (mean (SEM) 385.6 (26.1) arbitrary units, p < 0.01). A positive relation between CML and serum albumin (r = 0.394, p < 0.05) and lipofuscin (r = 0.651, p < 0.01) was found. CONCLUSIONS: The contrasting concentration of CML and AGE-Fl between patients with CHF and after heart transplantation in the presence of decreased GFR and oxidative stress are explained by lowered plasma proteins in CHF and higher concentrations in heart transplant recipients. In heart transplant recipients, in addition to myocardial inflammatory processes, immunosuppression may be important for enhanced formation of AGEs.

[Overweight and obesity--risk factors in the development and progression of renal disease]. / Nadváha a obezita--faktory podmienujúce vývin a progresiu ochorení obliciek.

Overweight/obesity represent an underestimated risk factor of renal disease. The incidence of obesity-related glomerulopathy (ORG) tremendously increased within the last decade. The first sign of renal damage in overweight conditions is microalbuminuria or proteinuria, indicating the potential risk of its progression to renal insufficiency and the development of premature cardiovascular events. In the early stage of obesity renal hemodynamics are characterized by a renal hypercirculation and glomerular hyperfiltration, particularly in the presence of hypertension. The hyperfiltration is especially harmful in patients with pre-existing inflammatory and metabolic renal disease, or under the conditions of reduced renal mass. Histopathologically, ORG is characterized by glomerulomegaly with/without signs of focal segmental glomerulosclerosis. Pathogenetically, numerous factors are involved, e.g. enhanced glomerular capillary pressure, adrenergic nerve overactivity, inappropriate activation of the renin-angiotensin-aldosterone system, insulin resistance, hyperinsulinemia and hyperleptinemia, dyslipidemia, enhanced clotting tendency and sodium retention. Diabetic nephropathy is one of the most serious complications of obesity-induced diabetes. In the industrial nations type 2 diabetes is the single most frequent cause of end-stage renal disease. After kidney transplantation, overweight/obesity is associated with a less favourable prognosis for the survival of the graft and the patient. Incidence of renal cell carcinomas is enhanced in overweight/obesity. Obesity-related renal disease may be prevented/postponed by an early weight reduction, by dietary intervention combined with physical exercise. In the advanced stages of renal disease benefits of weight reduction are minimal. Concomitant administration of angiotensin-converting-enzyme inhibitors or angiotensin II receptor 1 blockers exerts antiproteinuric effects and thereby aid in retarding the disease progression. Aimed prevention and treatment of obesity represent a challenge for the healthcare system. The concerted action of physicians, patients and the public health authorities is needed.