Studies on Matrix Metalloproteinase (MMP-2, -9 and -14) and β2 Integrin Targeting as Potential Anticancer Therapeutics

Proteolytic enzymes, such as matrix metalloproteinases (MMP), are associated to the progression of several cancers. They degrade extracellular components, which helps tumors to expand and cancer cells to escape from the primary site. Of all MMPs, gelatinases (MMP-2 and -9) and membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14), in particular, are often associated to more aggressive types of head and neck carcinomas as well as to a poorer outcome in patient survival. Although therapies during the last decades have advanced, the mortality of the disease is still rather high and adjuvant therapies are searched for continuously. MMP-9 and MT1-MMP are also involved in neo-angiogenesis, which is necessary for tumor expansion. For this reason, we have identified synthetic peptides-targeting gelatinases and MT1-MMP, and have also evaluated their anticancer effects in vitro and in vivo. Antigelatinolytic peptides effectively inhibited tongue-carcinoma cell invasion and reduced the growth of xenografted tumors. In tumor samples of mice that were treated with antigelatinolytic peptides, the micro-vessel density was significantly reduced. We also identified a novel MT1-MMP targeting peptide and demonstrated that it exerted anticancer effects against several malignant cell lines in vitro. The effects of MT1-MMP inhibition on tongue-squamous cell carcinomas were evaluated by using xenograft tumors, which it effectively inhibited. Tranexamic acid was also demonstrated to inhibit tongue-squamous cell carcinoma invasion, most probably due to its ability to prevent the plasmin-mediated activation of proMMP-9. Leukocyte β2 integrins are another interesting option when evaluating targets for the therapeutic intervention of inflammatory conditions or malignancies of hematopoietic origin, since β2 integrins are expressed mainly by leukocytes. We identified a novel technique for screening small-molecule libraries against β2 integrins, and by using this technique we identified a novel αMβ2 integrin-binding chemical (IMB-10). IMB-10 significantly enhances leukocyte adhesion and inhibits their motility. We also demonstrated that IMB-10 can be used to inhibit inflammation and lymphoma growth in vivo. Interestingly, IMB-10 also reduced leukocyte tumor infiltration and inhibited tumor invasion.

Oral Health among Young Adults and the Middle-aged in Iran

The aim of the present study was to assess oral health and treatment needs among adult Iranians according to socio-demographic status, smoking, and oral hygiene, and to investigate the relationships between these determinants and oral health. Data for 4448 young adult (aged 18) and 8301 middle-aged (aged 35 to 44) Iranians were collected in 2002 as part of a national survey using the World Health Organization (WHO) criteria for sampling and clinical diagnoses, across 28 provinces by 33 calibrated examiners. Gender, age, place of residence, and level of education served as socio-demographic information, smoking as behavioural and modified plaque index (PI) as the biological risk indicator for oral hygiene. Number of teeth, decayed teeth (DT), filled teeth (FT), decayed, missing, filled teeth (DMFT), community periodontal index (CPI), and prosthodontic rehabilitation served as outcome variables of oral health. Mean number of DMFT was 4.3 (Standard deviation (SD) = 3.7) in young adults and 11.0 (SD = 6.4) among middle-aged individuals. Among young adults the D-component (DT = 70%), and among middle-aged individuals the M-component (60%) dominated in the DMFT index. Among young adults, visible plaque was found in nearly all subjects. Maximum (max) PI was associated with higher mean number of DT, and higher periodontal treatment needs. A healthy periodontium was a rare condition, with 8% of young adults and 1% of middle-aged individuals having a max CPI = 0. The majority of the CPI findings among young adults consisted of calculus (48%) and deepened periodontal pockets (21%). Respective values for middle-aged individuals were 40% and 53%. Having a deep pocket (max CPI = 4) was more likely among young adults with a low level of education (Odds ratio (OR) = 2.7, 95% Confidence interval (CI) = 1.9–4.0) than it was among well-educated individuals. Among middle-aged individuals, having calculus or a periodontal pocket was more likely in men (OR = 1.8, 95% CI = 1.6–2.0) and in illiterate subjects (OR = 6.3, 95% CI = 5.1–7.8) than it was for their counterparts. Among young adults, having 28 teeth was more (p < 0.05) prevalent among men (72% vs. 68% for women), urban residents (71% vs. 67% for rural residents), and those with a high level of education (73% vs. 60% for those with a low level). Among middle-aged individuals, having a functional dentition was associated with younger age (OR = 2.0, 95% CI = 1.7−2.5) and higher level of education (OR = 1.8, 95% CI = 1.6−2.1). Of middle-aged individuals, 2% of 35- to 39-year-olds and 5% of those aged 40 to 44 were edentulous. Among the dentate subjects (n = 7,925), prosthodontic rehabilitation was more prevalent (p < 0.001) among women, urban residents, and those with a high level of education than it was among their counterparts. Among those having 1 to 19 teeth, a removable denture was the most common type of prosthodontic rehabilitation. Middle-aged individuals lacking a functional dentition were more likely (OR = 6.0, 95% CI = 4.8−7.6) to have prosthodontic rehabilitation than were those having a functional dentition. In total, 81% of all reported being non-smokers, and 32% of men and 5% of women were current smokers. Heavy smokers were the most likely to have deepened periodontal pockets (max CPI ≥ 3, OR = 2.9, 95% CI = 1.8−4.7) and to have less than 20 teeth (OR = 2.3, 95% CI = 1.5−3.6). The findings indicate impaired oral health status in adult Iranians, particularly those of low socio-economic status and educational level. The high prevalence of dental plaque and calculus and considerable unmet treatment needs call for a preventive population strategy with special emphasis on the improvement of oral self-care and smoking cessation to tackle the underlying risk factors for oral diseases in the Iranian adult population.

Candida proteinases in the degradation of oral mucosal tissue components associated with Candida invasion

The aim of this thesis was to compare the degradation of human oral epithelial proteins by proteinases of different Candida yeast species. We focused on proteins associated with Candida invasion in the cell-to-cell junction, the basement membrane zone, the extracellular matrix, and local tissue inflammatory regulators. Another main objective was to evaluate the effect of the yeast/hyphal transition and pH on the degradative capability of Candida. The enzymatic activity of the Candida proteinases was verified by gelatin zymography. Laminins-332 (Lm-322) and -511(Lm-511) produced by human oral keratinocytes were gathered from the growth media, and E-cadherin (E-Cad) was isolated from the cell membrane of the keratinocytes by immunoprecipitation. The proteins were incubated with Candida cells and cell-free fractions, and degradation was detected by fluorography. Fibronectin degradation was visualised by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Matrix metalloproteinase-9 (MMP-9) activation and tissue inhibitor of metalloproteinase-1 (TIMP-1) fragmentation was detected by using the Western blot and enhanced chemoluminescence (ECL) techniques. Residual activity of TIMP-1 was evaluated by a casein degradation assay. A fluorimetric assay was used to detect and compare Candida proteinase activities with MMP-9. These studies showed that the ability of the different Candida yeast species to degrade human Lm-332, fibronectin, and E-Cad vary from strain to strain and that this degradation is pH-dependent. This indicates that local acidic pH in tissue may play a role in tissue destruction by activating Candida proteinases and aid invasion of Candida into deeper tissue. A potential correlation exists between the morphological form of the yeasts and the degradative ability; the C. albicans yeast form seems to be related to superficial infections, and hyphal forms can apparently invade deeper tissues between the epithelial cells by degradation of E-Cad. Basement membrane degradation is possible, especially in the junctional epithelium, which contains only Lm-332 as a structural component. Local tissue host inflammatory mediators, such as MMP-9, were activated, and TIMP-1 was degraded by certain Candida species, thus indicating the possibility of a weakened host tissue defence mechanism in vivo.

Heparin versus 0.9% sodium chloride intermittent flushing for the prevention of occlusion in long term central venous catheters in infants and children: A systematic review

Background Around the world, guidelines and clinical practice for the prevention of complications associated with central venous catheters (CVC) vary greatly. To prevent occlusion, most institutions recommend the use of heparin when the CVC is not in use. However, there is debate regarding the need for heparin and evidence to suggest normal saline may be as effective. The use of heparin is not without risk, may be unnecessary and is also associated with increased costs. Objectives To assess the clinical effects (benefits and harms) of heparin versus normal saline to prevent occlusion in long-term central venous catheters in infants, children and adolescents. Design A Cochrane systematic review of randomised controlled trials was undertaken. - Data sources: The Cochrane Vascular Group Specialised Register (including MEDLINE, CINAHL, EMBASE and AMED) and the Cochrane Register of Studies were searched. Hand searching of relevant journals and reference lists of retrieved articles was also undertaken. - Review Methods: Data were extracted and appraisal undertaken. We included studies that compared the efficacy of normal saline with heparin to prevent occlusion. We excluded temporary CVCs and peripherally inserted central catheters. Rate ratios per 1000 catheter days were calculated for two outcomes, occlusion of the CVC, and CVC-associated blood stream infection. Results Three trials with a total of 245 participants were included in this review. The three trials directly compared the use of normal saline and heparin. However, between studies, all used different protocols with various concentrations of heparin and frequency of flushes. The quality of the evidence ranged from low to very low. The estimated rate ratio for CVC occlusion per 1000 catheter days between the normal saline and heparin group was 0.75 (95% CI 0.10 to 5.51, two studies, 229 participants, very low quality evidence). The estimated rate ratio for CVC-associated blood stream infection was 1.48 (95% CI 0.24 to 9.37, two studies, 231 participants; low quality evidence). Conclusions It remains unclear whether heparin is necessary for CVC maintenance. More well-designed studies are required to understand this relatively simple, but clinically important question. Ultimately, if this evidence were available, the development of evidenced-based clinical practice guidelines and consistency of practice would be facilitated.

Distinct subpopulations of gamma delta T cells are present in normal and tumor-bearing human liver

Gamma delta T cells are thought to mediate immune responses at epithelial surfaces. We have quantified and characterized hepatic and peripheral blood gamma delta T cells from 11 normal and 13 unresolved tumor-bearing human liver specimens. gamma delta T cells are enriched in normal liver (6.6% of T cells) relative to matched blood (0.9%; P = 0.008). The majority express CD4(-)CD8(-) phenotypes and many express CD56 and/or CD161. In vitro, hepatic gamma delta T cells can be induced to kill tumor cell lines and release interferon-gamma, tumor necrosis factor-alpha, interleukin-2 and interleukin-4. Analysis of V gamma and V delta chain usage indicated that V delta 3(+) cells are expanded in normal livers (21.2% of gamma delta T cells) compared to blood (0.5%; P = 0.001). Tumor-bearing livers had significant expansions and depletions of gamma delta T cell subsets but normal cytolytic activity. This study identifies novel populations of liver T cells that may play a role in immunity against tumors.

Origin of activation of Lattice Oxygen and Synergistic Interaction in Bimetal-Ionic Ce0.89Fe0.1Pd0.01O2-delta Catalyst

Flourite-type nanocrystalline Ce0.9Fe0.1O2-delta and Ce0.89Fe0.1Pd0.01O2-delta solid solutions have been synthesized by solution combustion method,'.which show higher oxygen storage/release property (OSC) compared to CeO2 and Ce0.8Zr0.2O2. Temperature programmed reduction an XPS study reveal that the presence of Pd ion in Ce0.9Fe0.1O2-delta facilitates complete reduction of Fe3+ to Fe2+ state and partial reduction of Ce4+ to Ce3+ state at.temperatures as low as 105 degrees C compared to 400 degrees C for monometal-ionic Ce0.9Fe0.1O2-delta. Fe3+ ion is reduced to Fe2+ and not to Feo due to favorable redox potential for Ce4+ + Fe2+ -> Ce3+ + Fe3+ reaction. Using first-principles density functional theory calculation we determine M-O (M = Pd, Fe, Ce) bond lengths, and find that bond lengths vary from shorter (2.16 angstrom) to longer (2.9 angstrom) bond distances compared to mean Ce-O bond distance of 2.34 angstrom. for CeO2. Using these results in bond valence analysis, we show that oxygen with bond valences as low as -1.55 are created, leading to activation of lattice oxygen in the bimetal ionic catalyst. Temperatures of CO oxidation and NO reduction by CO/H-2 are lower with the bimetalionic Ce0.89Fe0.1Pd0.01O2-delta catalyst compared to monometal-ionic Ce0.9Fe0.1O2-delta and Ce0.99Pd0.01O2-delta catalysts. From XPS studies of Pd impregnated on CeO2 and Fe2O3 oxides, we show that the synergism leading to low temperature activation of lattice oxygen in bimetal-ionic catalyst Ce0.89Fe0.1Pd0.01O2-delta is due to low-temperature reduction of Pd2+ to Pd-0, followed by Pd-0 + 2Fe(3+) -> Pd2+ + 2Fe(2+), Pd-0 + 2Ce(4+) -> Pd2+ + 2Ce(3+) redox reaction.

Study of sleep and evaluation of sumatriptan and rizatriptan in the acute treatment of migraine in children and adolescents

Migraine is a common disease in children and adolescents, affecting roughly 10% of school-aged children. Recent studies have revealed an increasing incidence of childhood migraine, but migraine remains an underrecognized and undertreated condition in the pediatric population. Migraine attacks are painful and disabling and can affect a child´s life in many ways. Effective drug treatment is usually needed. The new migraine drugs, triptans, were introduced at the beginning of the 1990s and have since been shown to be very effective in the treatment of migraine attacks in adults. Although they are widely used in adults, the acute treatment of migraine in children and adolescents is still based on paracetamol and nonsteroidal anti-inflammatory drugs. Some children can control their attacks satisfactorily with simple analgesics, but at least one-third need more powerful treatments. When this thesis work commenced, hardly any information existed on the efficacy and safety of triptans in children. The study aim of the thesis was to identify more efficient treatments of migraine for children and adolescents by investigating the efficacy of sumatriptan nasal spray and oral rizatriptan compared with placebo in them. Sleep has an impact on migraine in many aspects. Despite the clinical relevance and common manifestation of sleep in the context of migraine in children, very little research data on the true frequency of sleep exist. As sleeping is so often related to childhood migraine, it can be a confounding factor in clinical drug trials of migraine treatments in children and adolescents. How the results of a sleeping child should be analyzed is under continual debate. The aim of the thesis was also to clarify this as well as to evaluate the frequency of sleeping during migraine attacks in children and factors affecting frequency. Both nasal sumatriptan and oral rizatriptan were effective (superior to placebo), and well tolerated in treatment of migraine attacks in children and adolescents aged 8-17 and 6-17 years, respectively. No serous adverse effects were observed. The results of this work suggest that nasal sumatriptan 20 mg and rizatriptan 10 mg can be effectively and safely used to treat migraine attacks in adolescents aged over 12 years if more effective drugs than NSAIDs are needed. No difference was observed in efficacy or safety of nasal sumatriptan and rizatriptan between children aged younger than 12 years and older children, but because the treated number of patients under 12 years is still small, more studies are needed before sumatriptan or rizatriptan can be recommended for use in this population. Sleeping during migraine attacks was very common, and most children at least occasionally slept during an attack. Falling asleep was especially common in children under eight years of age and during the first hour after the onset of attack. Children who were able to sleep soon after attack onset were more likely pain-free at two hours. Sleeping probably both improves recovery from a migraine attack and is a sign of headache relief. Falling asleep should be classified as a sign of headache relief in clinical drug trials when studying migraine treatments in children and adolescents.

Inflammatory and coagulation disturbances in acute pancreatitis

Acute pancreatitis (AP), a common cause of acute abdominal pain, is usually a mild, self-limited disease. However, some 20-30% of patients develop a severe disease manifested by pancreatic necrosis, abscesses or pseudocysts, and/or extrapancreatic complications, such as vital organ failure (OF). Patients with AP develop systemic inflammation, which is considered to play a role in the pathogenesis of multiple organ failure (MOF). OF mimics the condition seen in patients with sepsis, which is characterized by an overwhelming production of inflammatory mediators, activation of the complement system and systemic activation of coagulation, as well as the development of disseminated intravascular coagulation (DIC) syndrome. Vital OF is the major cause of mortality in AP, along with infectious complications. About half of the deaths occur within the first week of hospitalization and thus, early identification of patients likely to develop OF is important. The aim of the present study was to investigate inflammatory and coagulation disturbances in AP and to find inflammatory and coagulation markers for predicting severe AP, and development of OF and fatal outcome. This clinical study consists of four parts. All of patients studied had AP when admitted to Helsinki University Central Hospital. In the first study, 31 patients with severe AP were investigated. Their plasma levels of protein C (PC) and activated protein C (APC), and monocyte HLA-DR expression were studied during the treatment period in the intensive care unit; 13 of these patients developed OF. In the second study, the serum levels of complement regulator protein CD59 were studied in 39 patients during the first week of hospitalization; 12 of them developed OF. In the third study, 165 patients were investigated; their plasma levels of soluble form of the receptor for advanced glycation end products (sRAGE) and high mobility group box 1 (HMGB1) protein were studied during the first 12 days of hos-pitalization; 38 developed OF. In the fourth study, 33 patients were studied on admission to hospital for plasma levels of prothrombin fragment F1+2 and tissue factor pathway inhibitor (TFPI), and thrombin formation capacity by calibrated automated thrombogram (CAT); 9 of them developed OF. Our results showed significant PC deficiency and decreased APC generation in patients with severe AP. The PC pathway defects seemed to be associated with the development of OF. In patients who developed OF, the levels of serum CD59 and plasma sRAGE, but not of HMGB1, were significantly higher than in patients who recovered without OF. The high CD59 levels on admission to the hospital seemed to be predictive for severe AP and OF. The median of the highest sRAGE levels was significantly higher in non-survivors than in survivors. No significant difference between the patient groups was found in the F1+2 levels. The thrombograms of all patients were disturbed in their shape, and in 11 patients the exogenous tissue factor did not trigger thrombin generation at all ( flat curve ). All of the patients that died displayed a flat curve. Free TFPI levels and free/total TFPI ratios were significantly higher in patients with a flat curve than in the others, and these levels were also significantly higher in non-survivors than in survivors. The flat curve in combination with free TFPI seemed to be predictive for a fatal outcome in AP.

Alveolar macrophages as accessory cells for human gamma delta T cells activated by Mycobacterium tuberculosis.

Alveolar macrophages form the first line of defense against inhaled droplets containing Mycobacterium tuberculosis by controlling mycobacterial growth and regulating T cell responses. CD4+ and gamma delta T cells, two major T cell subsets activated by M. tuberculosis, require accessory cells for activation. However, the ability of alveolar macrophages to function as accessory cells for T cell activation remains controversial. We sought to determine the ability of alveolar macrophages to serve as accessory cells for resting (HLA-DR-, IL-2R-) and activated (HLA-DR+, IL-2R+) gamma delta T cells in response to M. tuberculosis and its Ag, and to compare accessory cell function for gamma delta T cells of alveolar macrophages and blood monocytes obtained from the same donor. Alveolar macrophages were found to serve as accessory cells for both resting and activated gamma delta T cells in response to M. tuberculosis Ag. At high alveolar macrophage to T cell ratios (> 3:1), however, expansion of resting gamma delta T cells was inhibited by alveolar macrophages. The inhibition of resting gamma delta T cells by alveolar macrophages was dose-dependent, required their presence during the first 24 h, and was partially overcome by IL-2. Alveolar macrophages did not inhibit activated gamma delta T cells even at high accessory cell to T cell ratios, and alveolar macrophages functioned as well as monocytes as accessory cells. Monocytes were not inhibitory for either resting or activated gamma delta T cells. These findings support the following model. In the normal alveolus the alveolar macrophage to T cell ratio is > or = 9:1, and therefore the threshold for resting gamma delta T cell activation is likely to be high. Once a nonspecific inflammatory response occurs, such as after invasion by M. tuberculosis, this ratio is altered, favoring gamma delta T cell activation by alveolar macrophages.

Neurological manifestations and molecular genetics of acute intermittent porphyria in North Western Russia

Acute intermittent porphyria (AIP, MIM #176000) is an inherited metabolic disease due to a partial deficiency of the third enzyme, hydroxymethylbilane synthase (HMBS, EC: 4.3.1.8), in the haem biosynthesis. Neurological symptoms during an acute attack, which is the major manifestation of AIP, are variable and relatively rare, but may endanger a patient's life. In the present study, 12 Russian and two Finnish AIP patients with severe neurological manifestations during an acute attack were studied prospectively from 1995 to 2006. Autonomic neuropathy manifested as abdominal pain (88%), tachycardia (94%), hypertension (75%) and constipation (88%). The most common neurological sign was acute motor peripheral neuropathy (PNP, 81%) often associated with neuropathic sensory loss (54%) and CNS involvement (85%). Despite heterogeneity of the neurological manifestations in our patients with acute porphyria, the major pattern of PNP associated with abdominal pain, dysautonomia, CNS involvement and mild hepatopathy could be demonstrated. If more strict inclusion criteria for biochemical abnormalities (>10-fold increase in excretion of urinary PBG) are applied, neurological manifestations in an acute attack are probably more homogeneous than described previously, which suggests that some of the neurological patients described previously may not have acute porphyria but rather secondary porphyrinuria. Screening for acute porphyria using urinary PBG is useful in a selected group of neurological patients with acute PNP or encephalopathy and seizures associated with pain and dysautonomia. Clinical manifestations and the outcome of acute attacks were used as a basis for developing a 30-score scale of the severity of an acute attack. This scale can easily be used in clinical practice and to standardise the outcome of an attack. Degree of muscle weakness scored by MRC, prolonged mechanical ventilation, bulbar paralysis, impairment of consciousness and hyponatraemia were important signs of a poor prognosis. Arrhythmia was less important and autonomic dysfunction, severity of pain and mental symptoms did not affect the outcome. The delay in the diagnosis and repeated administrations of precipitating factors were the main cause of proceeding of an acute attack into pareses and severe CNS involvement and a fatal outcome in two patients. Nerve conduction studies and needle EMG were performed in eleven AIP patients during an acute attack and/or in remission. Nine patients had severe PNP and two patients had an acute encephalopathy but no clinically evident PNP. In addition to axonopathy, features suggestive of demyelination could be demonstrated in patients with severe PNP during an acute attack. PNP with a moderate muscle weakness was mainly pure axonal. Sensory involvement was common in acute PNP and could be subclinical. Decreased conduction velocities with normal amplitudes of evoked potentials during acute attacks with no clinically evident PNP indicated subclinical polyneuropathy. Reversible symmetrical lesions comparable with posterior reversible encephalopathy syndrome (PRES) were revealed in two patients' brain CT or MRI during an acute attack. In other five patients brain MRI during or soon after the symptoms was normal. The frequency of reversible brain oedema in AIP is probably under-estimated since it may be short-lasting and often indistinguishable on CT or MRI. In the present study, nine different mutations were identified in the HMBS gene in 11 unrelated Russian AIP patients from North Western Russia and their 32 relatives. AIP was diagnosed in nine symptom-free relatives. The majority of the mutations were family-specific and confirmed allelic heterogeneity also among Russian AIP patients. Three mutations, c.825+5G>C, c.825+3_825+6del and c.770T>C, were novel. Six mutations, c.77G>A (p.R26H), c.517C>T (p.R173W), c.583C>T (p.R195C), c.673C>T (p.R225X), c.739T>C (p.C247R) and c.748G>C (p.E250A), have previously been identified in AIP patients from Western and other Eastern European populations. The effects of novel mutations were studied by amplification and sequencing of the reverse-transcribed total RNA obtained from the patients' lymphoblastoid or fibroblast cell lines. The mutations c.825+5G>C and c.770T>C resulted in varyable amounts of abnormal transcripts, r.822_825del (p.C275fsX2) and [r.770u>c, r.652_771del, r.613_771del (p.L257P, p.G218_L257del, p.I205_L257del)]. All mutations demonstrated low residual activities (0.1-1.3 %) when expressed in COS-1 cells confirming the causality of the mutations and the enzymatic defect of the disease. The clinical outcome, prognosis and correlation between the HMBS genotype and phenotype were studied in 143 Finnish and Russian AIP patients with ten mutations (c.33G>T, c.97delA, InsAlu333, p.R149X, p.R167W, p.R173W, p.R173Q, p.R225G, p.R225X, c.1073delA) and more than six patients in each group. The patients were selected from the pool of 287 Finnish AIP patients presented in a Finnish Porphyria Register (1966-2003) and 23 Russian AIP patients (diagnosed 1995-2003). Patients with the p.R167W and p.R225G mutations showed lower penetrance (19% and 11%) and the recurrence rate (33% and 0%) in comparison to the patients with other mutations (range 36 to 67% and 0 to 66%, respectively), as well as milder biochemical abnormalities [urinary porphobilinogen 47±10 vs. 163±21 mol/L, p<0.001; uroporphyrin 130±40 vs. 942±183 nmol/L, p<0.001] suggesting a milder form of AIP in these patients. Erythrocyte HMBS activity did not correlate with the porphobilinogen excretion in remission or the clinical of the disease. In all AIP severity patients, normal PBG excretion predicted freedom from acute attacks. Urinary PBG excretion together with gender, age at the time of diagnosis and mutation type could predict the likelihood of acute attacks in AIP patients.

Complex trait of acute pancreatitis : Studies of candidate genes and adipokines

Acute pancreatitis (AP) is a common disease. Mild disease resolves spontaneously in a few days. Severe forms of the disease can lead to local complications, necrosis, and abscesses in and around the pancreas. Systemic inflammation in severe AP is associated with distant organ failures. The aim of this study is to identify genetically determined prognostic factors involved in the clinical features of AP. The study employs a candidate-gene approach, and the genes are involved in trysinogen activation in the initiation phase of the disease, as well as in the systemic inflammation as the disease proceeds. The last study examines adipokines, fat-derived hormones characterized with the capacity to modify inflammation. SPINK 1 is a gene coding trypsin activation inhibitor. Mutations N34S and P55N were determined by minisequencing methods in 371 AP patients and in 459 controls. The mutation N34S was more common in AP patients (7.8%) than in controls (2.6%). This suggests that SPINK 1 gene mutation N34S is a risk factor for AP. In the fourth study, in 12 matched pairs of patients with severe and mild AP, levels of adipokines, adiponectin, and leptin were evaluated. Plasma adipokine levels did not differ between patients with mild and severe AP. The results suggest that in AP, adipokine plasma levels are not factors predisposing to organ failures. This study identified the SPINK 1 mutation N34S to be a risk factor for AP in the general population. As AP is a multifactorial disease, and extensive genetic heterogeneity is likely, further identification of genetic factors in the disease requires larger future studies with more advanced genetic study models. Further identification of the patient characteristics associated with organ failures offers another direction of the study to achieve more detailed understanding of the severe form of AP.

Individualized immunosuppression after renal transplantation in childhood

Pediatric renal transplantation (TX) has evolved greatly during the past few decades, and today TX is considered the standard care for children with end-stage renal disease. In Finland, 191 children had received renal transplants by October 2007, and 42% of them have already reached adulthood. Improvements in treatment of end-stage renal disease, surgical techniques, intensive care medicine, and in immunosuppressive therapy have paved the way to the current highly successful outcomes of pediatric transplantation. In children, the transplanted graft should last for decades, and normal growth and development should be guaranteed. These objectives set considerable requirements in optimizing and fine-tuning the post-operative therapy. Careful optimization of immunosuppressive therapy is crucial in protecting the graft against rejection, but also in protecting the patient against adverse effects of the medication. In the present study, the results of a retrospective investigation into individualized dosing of immunosuppresive medication, based on pharmacokinetic profiles, therapeutic drug monitoring, graft function and histology studies, and glucocorticoid biological activity determinations, are reported. Subgroups of a total of 178 patients, who received renal transplants in 1988 2006 were included in the study. The mean age at TX was 6.5 years, and approximately 26% of the patients were <2 years of age. The most common diagnosis leading to renal TX was congenital nephrosis of the Finnish type (NPHS1). Pediatric patients in Finland receive standard triple immunosuppression consisting of cyclosporine A (CsA), methylprednisolone (MP) and azathioprine (AZA) after renal TX. Optimal dosing of these agents is important to prevent rejections and preserve graft function in one hand, and to avoid the potentially serious adverse effects on the other hand. CsA has a narrow therapeutic window and individually variable pharmacokinetics. Therapeutic monitoring of CsA is, therefore, mandatory. Traditionally, CsA monitoring has been based on pre-dose trough levels (C0), but recent pharmacokinetic and clinical studies have revealed that the immunosuppressive effect may be related to diurnal CsA exposure and blood CsA concentration 0-4 hours after dosing. The two-hour post-dose concentration (C2) has proved a reliable surrogate marker of CsA exposure. Individual starting doses of CsA were analyzed in 65 patients. A recommended dose based on a pre-TX pharmacokinetic study was calculated for each patient by the pre-TX protocol. The predicted dose was clearly higher in the youngest children than in the older ones (22.9±10.4 and 10.5±5.1 mg/kg/d in patients <2 and >8 years of age, respectively). The actually administered oral doses of CsA were collected for three weeks after TX and compared to the pharmacokinetically predicted dose. After the TX, dosing of CsA was adjusted according to clinical parameters and blood CsA trough concentration. The pharmacokinetically predicted dose and patient age were the two significant parameters explaining post-TX doses of CsA. Accordingly, young children received significantly higher oral doses of CsA than the older ones. The correlation to the actually administered doses after TX was best in those patients, who had a predicted dose clearly higher or lower (> ±25%) than the average in their age-group. Due to the great individual variation in pharmacokinetics standardized dosing of CsA (based on body mass or surface area) may not be adequate. Pre-Tx profiles are helpful in determining suitable initial CsA doses. CsA monitoring based on trough and C2 concentrations was analyzed in 47 patients, who received renal transplants in 2001 2006. C0, C2 and experienced acute rejections were collected during the post-TX hospitalization, and also three months after TX when the first protocol core biopsy was obtained. The patients who remained rejection free had slightly higher C2 concentrations, especially very early after TX. However, after the first two weeks also the trough level was higher in the rejection-free patients than in those with acute rejections. Three months after TX the trough level was higher in patients with normal histology than in those with rejection changes in the routine biopsy. Monitoring of both the trough level and C2 may thus be warranted to guarantee sufficient peak concentration and baseline immunosuppression on one hand and to avoid over-exposure on the other hand. Controlling of rejection in the early months after transplantation is crucial as it may contribute to the development of long-term allograft nephropathy. Recently, it has become evident that immunoactivation fulfilling the histological criteria of acute rejection is possible in a well functioning graft with no clinical sings or laboratory perturbations. The influence of treatment of subclinical rejection, diagnosed in 3-month protocol biopsy, to graft function and histology 18 months after TX was analyzed in 22 patients and compared to 35 historical control patients. The incidence of subclinical rejection at three months was 43%, and the patients received a standard rejection treatment (a course of increased MP) and/or increased baseline immunosuppression, depending on the severity of rejection and graft function. Glomerular filtration rate (GFR) at 18 months was significantly better in the patients who were screened and treated for subclinical rejection in comparison to the historical patients (86.7±22.5 vs. 67.9±31.9 ml/min/1.73m2, respectively). The improvement was most remarkable in the youngest (<2 years) age group (94.1±11.0 vs. 67.9±26.8 ml/min/1.73m2). Histological findings of chronic allograft nephropathy were also more common in the historical patients in the 18-month protocol biopsy. All pediatric renal TX patients receive MP as a part of the baseline immunosuppression. Although the maintenance dose of MP is very low in the majority of the patients, the well-known steroid-related adverse affects are not uncommon. It has been shown in a previous study in Finnish pediatric TX patients that steroid exposure, measured as area under concentration-time curve (AUC), rather than the dose correlates with the adverse effects. In the present study, MP AUC was measured in sixteen stable maintenance patients, and a correlation with excess weight gain during 12 months after TX as well as with height deficit was found. A novel bioassay measuring the activation of glucocorticoid receptor dependent transcription cascade was also employed to assess the biological effect of MP. Glucocorticoid bioactivity was found to be related to the adverse effects, although the relationship was not as apparent as that with serum MP concentration. The findings in this study support individualized monitoring and adjustment of immunosuppression based on pharmacokinetics, graft function and histology. Pharmacokinetic profiles are helpful in estimating drug exposure and thus identifying the patients who might be at risk for excessive or insufficient immunosuppression. Individualized doses and monitoring of blood concentrations should definitely be employed with CsA, but possibly also with steroids. As an alternative to complete steroid withdrawal, individualized dosing based on drug exposure monitoring might help in avoiding the adverse effects. Early screening and treatment of subclinical immunoactivation is beneficial as it improves the prospects of good long-term graft function.