Responses of Scots pine to nickel and copper exposure and herbivory

The goal of this thesis was to examine the ecophysiological responses of Scots pine (Pinus sylvestris L.), with an emphasis on the oxidative enzyme peroxidase and plant phenolics to environmental stresses like elevated levels of nickel (Ni) and copper (Cu), and herbivory. The effects of Ni and Cu were studied in a gradient survey at a sulphur dioxide contaminated site in the Kola Peninsula, and with experiments in which seedlings were exposed to Ni mist or to Ni and Cu amended into the soil. In addition, experimental Ni exposure was combined with disturbance of the natural lichen cover of the forest ground layer. Pine sawfly attack was simulated in the early season defoliation experiment, in which mature Scots pine were defoliated (100 %) during two successive years in a dry, nutrient-poor Scots pine stand. In addition, the effect of previous defoliation on the growth of sawfly (Diprion pini L.) larvae was studied. Apoplastic peroxidase activity was elevated in the needles of pine in a Ni- , Cu- and SO2- polluted environment, which indicated an increased oxidative stress. Increased foliar peroxidase activity due to Ni contamination was shown in the experiment, in which Ni was added as mist. No such response was found in peroxidase acitivity of the roots exposed to elevated Ni and/or Cu in the soil. Elevated Ni in the soil increased the concentration of foliar condensed tannins, which are able to bind heavy metals in the cells. Addition of low levels of Ni in the soil appeared to benefit pine seedlings, which was seen as promoted shoot growth and better condition of the roots. Wet Ni deposition of 2000 mg m-2 reduced growth and survival of pine seedlings, whereas deposition levels 200 mg m-2 or 20 mg m-2 caused no effects in a 2-y lasting experiment. The lichen mat on the forest floor did not act as an effective buffer against the adverse impacts of heavy metals on pine seedlings. However, some evidence was found indicating that soil microbes profited from the lichen mat. Artificial defoliation increased peroxidase activity in the Scots pine needles. In addition, defoliation decreased nitrogen, diamine putrescine and glucose concentrations in the needles and increased the concentrations of several phenolic compounds, starch and sucrose. Previous artificial defoliation led to poor growth of sawfly larvae reared on the pines, suggesting delayed induced resistance in Scots pine. However, there was no consistent relationship between inducibility (proportional increase in a compound following defoliation) and adverse effects on the growth of pine sawfly larvae. The observed inducible responses in needle phenolics due to previous defoliation thus appear to represent non-specific responses against sawflies.

Genetic aspects of outcome in kidney transplantation: cytokine and thrombosis associated candidate genes and gene expression biomarkers

Kidney transplantation (Tx) is the treatment of choice for end stage renal disease. Immunosuppressive medications are given to prevent an immunological rejection of the transplant. However, immunosuppressive drugs increase e.g. the risk of infection, cancer or nephrotoxicity. A major genetic contributors to immunological acceptance of the graft are human leukocyte antigen (HLA) genes. Also other non-HLA gene polymorphisms may predict the future risk of complications before Tx, possibly enabling individualised immunotherapy. Graft function after Tx is monitored using non-specific clinical symptoms and laboratory markers. The definitive diagnosis of graft rejection however relies on a biopsy of the graft. In the acute rejection (AR) diagnostics there is a need for an alternative to biopsy that would be an easily repeatable and simple method for regular use. Frequent surveillance of acute or subclinical rejection (SCR) may improve long-term function. In this thesis, associations between cytokine and thrombosis associated candidate genes and the outcome of kidney Tx were studied. Cytotoxic and co-stimulatory T lymphocyte molecule gene expression biomarkers for the diagnosis of the AR and the SCR were also investigated. We found that polymorphisms in the cytokine genes tumor necrosis factor and interleukin 10 (IL10) of the recipients were associated with AR. In addition, certain IL10 gene polymorphisms of the donors were associated with the incidence of cytomegalovirus infection and occurrence of later infection in a subpopulation of recipients. Further, polymorphisms in genes related to the risk of thrombosis and those of certain cytokines were not associated with the occurrence of thrombosis, infarction, AR or graft survival. In the study of biomarkers for AR, whole blood samples were prospectively collected from adult kidney Tx patients. With real-time quantitative PCR (RT-QPCR) gene expression quantities of CD154 and ICOS differentiated the patients with AR from those without, but not from the patients with other causes of graft dysfunction. Biomarkers for SCR were studied in paediatric kidney Tx patients. We used RT-QPCR to quantify the gene expression of immunological candidate genes in a low-density array format. In addition, we used RT-QPCR to validate the results of the microarray analysis. No gene marker differentiated patients with SCR from those without SCR. This research demonstrates the lack of robust markers among polymorphisms or biomarkers in investigated genes that could be included in routine analysis in a clinical laboratory. In genetic studies, kidney Tx can be regarded as a complex trait, i.e. several environmental and genetic factors may determine its outcome. A number of currently unknown genetic factors probably influence the results of Tx.

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation Loss of facial symmetry and mimetic function as seen in facial paralysis has an enormous impact on the psychosocial conditions of the patients. Patients with severe long-term facial paralysis are often reanimated with a two-stage procedure combining cross-facial nerve grafting, and 6 to 8 months later with microneurovascular (MNV) muscle transfer. In this thesis, we recorded the long-term results of MNV surgery in facial paralysis and observed the possible contributing factors to final functional and aesthetic outcome after this procedure. Twenty-seven out of forty patients operated on were interviewed, and the functional outcome was graded. Magnetic resonance imaging (MRI) of MNV muscle flaps was done, and nerve graft samples (n=37) were obtained in second stage of the operation and muscle biopsies (n=18) were taken during secondary operations.. The structure of MNV muscles and nerve grafts was evaluated using histological and immunohistochemical methods ( Ki-67, anti-myosin fast, S-100, NF-200, CD-31, p75NGFR, VEGF, Flt-1, Flk-1). Statistical analysis was performed. In our studies, we found that almost two-thirds of the patients achieved good result in facial reanimation. The longer the follow-up time after muscle transfer the weaker was the muscle function. A majority of the patients (78%) defined their quality of life improved after surgery. In MRI study, the free MNV flaps were significantly smaller than originally. A correlation was found between good functional outcome and normal muscle structure in MRI. In muscle biopsies, the mean muscle fiber diameter was diminished to 40% compared to control values. Proliferative activity of satellite cells was seen in 60% of the samples and it tended to decline with an increase of follow-up time. All samples showed intramuscular innervation. Severe muscle atrophy correlated with prolonged intraoperative ischaemia. The good long-term functional outcome correlated with dominance of fast fibers in muscle grafts. In nerve grafts, the mean number of viable axons amounted to 38% of that in control samples. The grafted nerves characterized by fibrosis and regenerated axons were thinner than in control samples although they were well vascularized. A longer time between cross facial nerve grafting and biopsy sampling correlated with a higher number of viable axons. P75Nerve Growth Factor Receptor (p75NGFR) was expressed in every nerve graft sample. The expression of p75NGFR was lower in older than in younger patients. A high expression of p75NGFR was often seen with better function of the transplanted muscle. In grafted nerve Vascular Endothelial Growth Factor (VEGF) and its receptors were expressed in nervous tissue. In conclusion, most of the patients achieved good result in facial reanimation and were satisfied with the functional outcome. The mimic function was poorer in patients with longer follow-up time. MRI can be used to evaluate the structure of the microneurovascular muscle flaps. Regeneration of the muscle flaps was still going on many years after the transplantation and reinnervation was seen in all muscle samples. Grafted nerves were characterized by fibrosis and fewer, thinner axons compared to control nerves although they were well vascularized. P75NGFR and VEGF were expressed in human nerve grafts with higher intensity than in control nerves which is described for the first time.

Evaluation of regional and distant metastases in head and neck squamous cell carcinoma patients with special reference to the assessment of regional lymph nodes in oral cancer

For optimal treatment planning, a thorough assessment of the metastatic status of mucosal squamous cell carcinoma of the head and neck (HNSCC) is required. Current imaging methods do not allow the recognition of all patients with metastatic disease. Therefore, elective treatment of the cervical lymph nodes is usually given to patients in whom the risk of subclinical metastasis is estimated to exceed 15-20%. The objective of this study was to improve the pre-treatment evaluation of patients diagnosed with HNSCC. Particularly, we aimed at improving the identification of patients who will benefit from elective neck treatment. Computed tomography (CT) of the chest and abdomen was performed prospectively for 100 patients diagnosed with HNSCC. The findings were analysed to clarify the indications for this examination in this patient group. CT of the chest influenced the treatment approach in 3% of patients, while CT of the abdomen did not reveal any significant findings. Our results suggest that CT of the chest and abdomen is not indicated routinely for patients with newly diagnosed HNSCC but can be considered in selected cases. Retrospective analysis of 80 patients treated for early stage squamous cell carcinoma of the oral tongue was performed to investigate the potential benefits of elective neck treatment and to examine whether histopathological features of the primary tumour could be used in the prediction of occult metastases, local recurrence, or/and poor survival. Patients who had received elective neck treatment had significantly fewer cervical recurrences during the follow-up when compared to those who only had close observation of the cervical lymph nodes. Elective neck treatment did not result in survival benefit, however. Of the histopathological parameters examined, depth of infiltration and pT-category (representing tumour diameter) predicted occult cervical metastasis, but only the pT-category predicted local recurrence. Depth of infiltration can be used in the identification of at risk patients but no clear cut-off value separating high-risk and low-risk patients was found. None of the histopathological parameters examined predicted survival. Sentinel lymph node (SLN) biopsy was studied as a means of diagnosing patients with subclinical cervical metastases. SLN biopsy was applied to 46 patients who underwent elective neck dissection for oral squamous cell carcinoma. In addition, SLN biopsy was applied to 13 patients with small oral cavity tumours who were not intended to undergo elective neck dissection because of low risk of occult metastasis. The sensitivity of SLN biopsy for finding subclinical cervical metastases was found to be 67%, when SLN status was compared to the metastatic status of the rest of the neck dissection specimen. Of the patients not planned to have elective neck dissection, SLN biopsy revealed cervical metastasis in 15% of the patients. Our results suggest that SLN biopsy can not yet entirely replace elective neck dissection in the treatment of oral cancer, but it seems beneficial for patients with low risk of metastasis who are not intended for elective neck treatment according to current treatment protocols.

Noninvasive monitoring of activity in Crohn's disease

Background: The fecal neutrophil-derived proteins calprotectin and lactoferrin have proven useful surrogate markers of intestinal inflammation. The aim of this study was to compare fecal calprotectin and lactoferrin concentrations to clinically, endoscopically, and histologically assessed Crohn’s disease (CD) activity, and to explore the suitability of these proteins as surrogate markers of mucosal healing during anti-TNFα therapy. Furthermore, we studied changes in the number and expression of effector and regulatory T cells in bowel biopsy specimens during anti-TNFα therapy. Patients and methods: Adult CD patients referred for ileocolonoscopy (n=106 for 77 patients) for various reasons were recruited (Study I). Clinical disease activity was assessed with the Crohn’s disease activity index (CDAI) and endoscopic activity with both the Crohn’s disease index of severity (CDEIS) and the simple endoscopic score for Crohn’s disease (SES-CD). Stool samples for measurements of calprotectin and lactoferrin, and blood samples for CRP were collected. For Study II, biopsy specimens were obtained from the ileum and the colon for histologic activity scoring. In prospective Study III, after baseline ileocolonoscopy, 15 patients received induction with anti-TNFα blocking agents and endoscopic, histologic, and fecal-marker responses to therapy were evaluated at 12 weeks. For detecting changes in the number and expression of effector and regulatory T cells, biopsy specimens were taken from the most severely diseased lesions in the ileum and the colon (Study IV). Results: Endoscopic scores correlated significantly with fecal calprotectin and lactoferrin (p<0.001). Both fecal markers were significantly lower in patients with endoscopically inactive than with active disease (p<0.001). In detecting endoscopically active disease, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for calprotectin ≥200 μg/g were 70%, 92%, 94%, and 61%; for lactoferrin ≥10 μg/g they were 66%, 92%, 94%, and 59%. Accordingly, the sensitivity, specificity, PPV, and NPV for CRP >5 mg/l were 48%, 91%, 91%, and 48%. Fecal markers were significantly higher in active colonic (both p<0.001) or ileocolonic (calprotectin p=0.028, lactoferrin p=0.004) than in ileal disease. In ileocolonic or colonic disease, colon histology score correlated significantly with fecal calprotectin (r=0.563) and lactoferrin (r=0.543). In patients receiving anti-TNFα therapy, median fecal calprotectin decreased from 1173 μg/g (range 88-15326) to 130 μg/g (13-1419) and lactoferrin from 105.0 μg/g (4.2-1258.9) to 2.7 μg/g (0.0-228.5), both p=0.001. The relation of ileal IL-17+ cells to CD4+ cells decreased significantly during anti-TNF treatment (p=0.047). The relation of IL-17+ cells to Foxp3+ cells was higher in the patients’ baseline specimens than in their post-treatment specimens (p=0.038). Conclusions: For evaluation of CD activity, based on endoscopic findings, more sensitive surrogate markers than CDAI and CRP were fecal calprotectin and lactoferrin. Fecal calprotectin and lactoferrin were significantly higher in endoscopically active disease than in endoscopic remission. In both ileocolonic and colonic disease, fecal markers correlated closely with histologic disease activity. In CD, these neutrophil-derived proteins thus seem to be useful surrogate markers of endoscopic activity. During anti-TNFα therapy, fecal calprotectin and lactoferrin decreased significantly. The anti-TNFα treatment was also reflected in a decreased IL-17/Foxp3 cell ratio, which may indicate improved balance between effector and regulatory T cells with treatment.

Surveillance in hereditary nonpolyposis colorectal cancer syndrome

Hereditary nonpolyposis colorectal cancer (HNPCC) is an inherited cancer predisposition syn-drome characterized by early onset colorectal cancer (CRC) and several other extra-colonic cancers, most commonly endometrial cancer (EC) and gastric cancer. Our aim was to evaluate the efficiency and results of the ongoing CRC and EC surveillance programs and to investigate the grounds for future gastric cancer screening by comparing the gastric biopsies of mutation positive and negative siblings in search for premalignant lesions. We also compared a new surveillance method, computerized tomographic colonoscopy (CTC) with optic colonoscopy. The patient material consisted of 579 family members from 111 Finnish HNPCC families al-most all harboring a known mismatch repair gene mutation. The efficacy of CRC and EC surveillance programs on HNPCC patients was evaluated by comparing the stage and survival of cancer cases detected with surveillance versus without. The performance of a new technique, CTC, was explored using a same-day colonoscopy as a reference standard. The use of intrauterine aspiration biopsies for EC surveillance was intro-duced for the first time in a HNPCC setting. Upper GI endoscopies were performed and biop-sies taken from mutation carriers and their mutation-negative siblings. The present surveillance program for CRC proved to be efficient. The CRC cases detected by surveillance were at a significantly more favorable stage than those in the non-surveilled group. This advantage was reflected in a significantly higher CRC-specific survival in the sur-veilled group. The performance of a new technique, CTC was found insufficient for polyp detection in this population in which every polyp, no matter the size, should be detected and removed. Colono-scopy was confirmed as a better surveillance modality than CTC. We could not observe any of the assumed differences in the gastric mucosa from mutation carriers and their mutation-negative siblings and no cases of gastric cancer were detected. The results gave no support for gastric surveillance. The EC surveillance program (transvaginal ultrasound and intra-uterine biopsy every 2-3 years) seemed to be efficient. It yielded several asymptomatic cancer cases and premalignant lesions. The stage distribution of the endometrial cancers in the group under surveillance tended to be more favorable than that of the mutation-positive, symptomatic EC patients who had no surveillance. None of the surveilled EC patients died of EC compared to six in the non-surveilled patients during the follow up. The improvement was, however, not statistically sig-nificant, thus far. Another observation was the good performance of endometrial aspiration biopsies used in this setting for the first time.

Immediate Breast Reconstructions in the Treatment of Breast Cancer

Breast cancer is the most common form of potentially fatal cancer in women in the Western world. Better understanding of the breast cancer disease process together with developments in treatments have led to improved survival and reduced risk of recurrence, significantly influencing the acceptance of breast reconstructions as part of breast cancer treatment. Skin-sparing mastectomy followed by immediate breast reconstruction has proved superior to other forms of breast reconstruction in terms of aesthetic outcome. However, due to the relatively recent introduction of skin-sparing mastectomy concerns on the surgical and oncological safety of the operation persist. The aim of the present study is to evaluate the surgical and oncological safety of skin-sparing mastectomy and immediate breast reconstruction in a consecutive patient series with ensuing follow-up. Subsequent aims of the study are to examine possibilities of reducing surgical complications of the operation and to assess the feasibility of sentinel node biopsy together with immediate breast reconstruction. The study population comprises a consecutive series of patients having undergone skin-sparing mastectomy followed by immediate breast reconstruction at the Helsinki University Central Hospital between 1992 and 2006. In Study I, the hospital records of 207 patients, operated between 1992 and 2001, were analyzed for surgical complications and recurrences of breast cancer during follow-up. In Study II, 60 consecutive patients were randomized into either conventional diathermy or radiofrequency coagulation groups to examine possibilities of reducing skin-flap complications. Study III consists of 62 consecutive breast cancer patients evaluated for the feasibility of sentinel node biopsy simultaneously with immediate breast reconstruction. In Study IV, hospital records were analyzed to examine local recurrence of breast cancer in a consecutive series of 146 patients with Stage I or II disease. Post-operative complications in Study I included native skin-flap necrosis (10.1%), hematoma (10.1%), anastomose thrombosis (5.3%), infection (3.4%), hernia (2.6%) and loss of one microvascular flap (0.7%). The Stage I and II patients in Study IV had a local recurrence rate of 2.7%, an isolated regional lymph node recurrence rate of 2.1% and a systemic recurrence rate of 2.7%, during a mean follow-up time of 51 months. The Stage III patients in study I had a locoregional recurrence rate of 31.3% during follow-up. Radiofrequency coagulation in Study II did not decrease skin-flap complications when compared with conventional diathermy. An increased skin-flap complication rate in Study II was associated with smoking and the type of skin incision used. In Study III, eleven patients had tumor positive sentinel nodes, nine of which were detected intraoperatively. Skin-sparing mastectomy followed by immediate breast reconstruction is a safe procedure both surgically and oncologically, especially for early stage breast cancer. Tennis racket type incision is associated with an increased skin-flap complication rate. Sentinel node biopsy with intraoperative assessment of sentinel node metastases is feasible in patients undergoing immediate breast reconstruction.

Epidemiology and Treatment Options of Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is caused by an autoimmune inflammation of the small bile ducts. It results to destruction of bile ducts, accumulation of the bile in the liver, and cirrhosis. The prevalence and incidence of PBC is increasing in the Western world. The prevalence is highest in the USA (402 per million) and incidence in Scotland (49/million/year). Our aim was to assess the epidemiology of PBC in Finland. Patients for the epidemiological study were searched from the hospital discharge records from year 1988 to 1999.The prevalence rose from 103 to 180/million from 1988 to 1999, an annual increase of 5.1%. The incidence rose from 12 to 17 /million/year, an annual increase of 3.5%. The age at death increased markedly from 65 to 76 years. The risk of liver related deaths diminished over time. The treatment of PBC is based on Ursodeoxycholic acid (UDCA). During 20 years 50% of patients end up with cirrhosis. Our treatment option was to combine budesonide, a potent corticosteroid with a high first pass metabolism in the liver, to UDCA and evaluate the liver effects and systemic effects such as bone mass density (BMD) changes. Our aim was to find out if combination of laboratory tests would serve as a surrogate marker for PBC and help reducing the need for liver biopsy. Non-cirrhotic PBC patients were randomized to receive budesonide 6 mg/day combined to UDCA 15 mg /kg/day or UDCA alone for three years. The combination therapy with UDCA and budesonide was effective: stage improved 22%, fibrosis 25%, and inflammation 32%. In the UDCA group the changes were: 20% deterioriation in stage and 70% in fibrosis, but a 10% improvement in inflammation. BMD in femoral neck decreased by 3.6% in the combination group and by 1.9% in the UDCA group. The reductions in lumbar spine were 2.8% and 0.7%. Pharmacokinetics did not differ between the stages of PBC. HA, PIIINP, bile acids, and AST were significantly different within stages I-III and could differentiate the mild fibrosis (F0F1) from the moderate (F2F3). The combination of these individual markers (PBC-score) further improved the accuracy. The area under the ROC of the PBC score, using a cut of value 66, had a sensitivity of 81.4% and a specificity of 65.2% to classify the stage of PBC. The prevalence of PBC in Finland increases, which results from increasing incidence and improved survival. The combination of budesonide and UDCA improves liver histology compared to UDCA alone in non-cirrhotic stages of PBC. The treatment may reduce BMD. Hyaluronic acid, PIIINP, AST, and bile acids may serve as tools to monitor the treatment response in the early stages of PBC. The budesonide and UDCA combination therapy is an option for those patients who do not receive full response from UDCA and are still at the non-cirrhotic stage of PBC.

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.

Herpes Simplex Virus Infection, Pathological Pain and Recurrent Lymphocytic Meningitis

Background: Aims of the study were: (i) to characterise the clinical picture, immunological features and changes in brain morphology and function in patients with widespread unilateral pain and HSV-infections, and (ii) to analyse the prevalence, clinical symptoms and immunological predisposing factors of HSV-2 induced recurrent lymphocytic meningitis (RLM) in Southern Finland. Patients and methods: Patients for the studies were recruited from the Pain Clinic, and from the Department of Neurology, at Helsinki University Central Hospital. Plasma concentrations of IgM, IgA, IgG, and IgG1-4, and serum concentrations of C3, C4 were measured. Serological anti-HSV-1 and -2 antibody status was tested. C4 genotyping, HLA-A, HLA-B and HLA-DRB1 typing, MBL2 genotyping, and IgG1 and IgG3 allotyping (Gm) were performed. Clinical neurological examination, quantitative sensory testing, skin biopsy, and functional magnetic resonance imaging were also performed. Results: HSV probably has a role in the generation of a pathological pain state. Low serum IgG1 and IgG3 levels, made the patients vulnerable for recurring HSV infections. Both functional and structural changes were observed in the brain pain-processing areas in the patients: they had less pain-related activity in the insular cortices bilaterally, in the anterior cingular cortex (ACC), and in the thalamus, and the gray matter density was lower in the ACC, in the frontal and prefrontal cortices. In the meningitis studies it was shown that RLM is more common and less benign than previously reported, and that neuropathic pain is frequently present both during and after meningitis episodes. HLA-DRB1*01, HLA-B*27, and low IgG1 levels are predisposing factors for RLM. Conclusions: Patients are vulnerable to recurrent HSV infections because of subtle immunological abnormalities. HSV causes diverse clinical manifestations. First, the herpes simplex virus, or the inflammatory process triggered by it, may cause pathological widespread pain probably by activating glial cells in the CNS. In these patients, signs of alterations in the brain pain-processing areas can be demonstrated by functional brain imaging methods. Secondly, HSV-2 induced RLM is a rare complication of HSV-2 virus. The predisposing factors include low IgG1 subclass levels, HLA-DRB1*01 and HLA –B*27 genotypes. Neuropathic pain is frequently associated with RLM.

Conjunctival Melanoma in Finland 1967-2000: a population-based study

Eighty-five new cases of conjunctival melanoma (CM) were diagnosed in Finland between 1967 and 2000. The annual crude incidence of CM was 0.51 per million inhabitants. The average age-adjusted incidence of 0.54 doubled during the study period, analogous to the increase in the incidence of cutaneous malignant melanoma during this period, suggesting a possible role for ultraviolet radiation in its pathogenesis. Nonlimbal tumors were more likely than limbal ones to recur and they were associated with decreased survival. Increasing tumor thickness and recurrence of the primary tumor were other clinical factors related to death from CM. The histopathologic specimens of 85 patients with CM melanoma were studied for cell type, mitotic count, tumor-infiltrating lymphocytes and macrophages, mean vascular density, extravascular matrix loops and networks, and mean diameter of the ten largest nucleoli (MLN). The absence of epithelioid cells, increasing mitotic count and small MLN were associated with shorter time to recurrence according to the Cox univariate regression. None of the histopathologic variables was associated with mortality from CM. Four (5%) patients had a CM limited to the cornea without evidence of a tumor other than primary acquired melanosis of the conjunctiva. Because there are no melanocytes in the cornea, the origin of these melanomas most likely is the limbal conjunctiva. All four corneally displaced CM were limited to the epithelium, and none of the patients developed metastases. An anatomic sub-classification based on my patients and world literature was developed for corneally displaced CM. In 20 patients the metastatic pattern could be determined. Ten patients had initial systemic metastases detected, nine had initial regional metastases, and in one case the two types were detected simultaneously. The patients most likely to develop either type of initial metastases were those with nonlimbal conjunctival melanoma, those with a primary tumor more than 2 mm thick, and those with a recurrent conjunctival melanoma. Approximately two thirds of the patients had limbal CM, a location associated with good prognosis. One third, however, had a primary CM originating outside the limbus. In these patients the chance of developing local recurrences as well as systemic metastases was significantly higher than in patients with limbal CM. Each recurrence accompanies an increased risk of developing metastases, and recurrences contribute to death along with increasing tumor thickness and nonlimbal tumor location. In my data, an equal number of patients with initial locoregional and systemic metastasis existed. Patients with limbal primary tumors less than 2 mm in thickness rarely experienced metastases, unless the tumor recurred. Consequently, the patients most likely to benefit from sentinel lymph node biopsy are those who have nonlimbal tumors, CM that are over 2 mm thick, or recurrent CM. The histopathology of CM differs from that of uveal melanoma. Microvascular factors did not prove to be of prognostic importance, possibly due to the fact that CM at least as often disseminates first to the regional lymph nodes, unlike uveal melanoma that almost always disseminates hematogenously.

Testicular function in adolescent boys with Klinefelter syndrome

Klinefelter syndrome (KS) is the most frequent karyotype disorder of male reproductive function. Since its original clinical description in 1942 and the identification of its chromosomal basis 47,XXY in 1959, the typical KS phenotype has become well recognized, but the mechanisms behind the testicular degeneration process have remained unrevealed. This prospective study was undertaken to increase knowledge about testicular function in adolescent KS boys. It comprised a longitudinal follow-up of growth, pubertal development, and serum reproductive hormone levels in 14 prepubertal and pubertal KS boys. Each boy had a testicular biopsy that was analyzed with histomorphometric and immunohistochemical methods. The KS boys had sufficient testosterone levels to allow normal onset and progression of puberty. Their serum testosterone levels remained within the low-normal range throughout puberty, but from midpuberty onwards, findings like a leveling-off in testosterone and insulin-like factor 3 (INSL3) concentrations, high gonadotropin levels, and exaggerated responses to gonadotropin-releasing hormone stimulation suggest diminished testosterone secretion. We also showed that the Leydig cell differentiation marker INSL3 may serve as a novel marker for onset and normal progression of puberty in boys. In the KS boys the number of germ cells was already markedly lower at the onset of puberty. The pubertal activation of the pituitary-testicular axis accelerated germ cell depletion, and germ cell differentiation was at least partly blocked at the spermatogonium or early primary spermatocyte stages. The presence of germ cells correlated with serum reproductive hormone levels. The immature Sertoli cells were incapable of transforming to the adult type, and during puberty the degeneration of Sertoli cells increased markedly. The older KS boys displayed an evident Leydig cell hyperplasia, as well as fibrosis and hyalinization of the interstitium and peritubular connective tissue. Altered immunoexpression of the androgen receptor (AR) suggested that in KS boys during puberty a relative androgen deficiency develops at testicular level. The impact of genetic features of the supernumerary X chromosome on the KS phenotype was also studied. The present study suggests that parental origin of the supernumerary X chromosome and the length of the CAG repeat of the AR gene influence pubertal development and testicular degeneration. The current study characterized by several means the testicular degeneration process in the testes of adolescent KS boys and confirmed that this process accelerates at the onset of puberty. Although serum reproductive hormone levels indicated no hypogonadism during early puberty, the histological analyses showed an already markedly reduced fertility potential in prepubertal KS boys. Genetic features of the X chromosome affect the KS phenotype.

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.

IgA nephropathy and Henoch-Schönlein nephritis in adults : with special reference to factors affecting outcome

IgA nephropathy (IgAN) is the most common primary glomerulonephritis. In one third of the patients the disease progresses, and they eventually need renal replacement therapy. IgAN is in most cases a slowly progressing disease, and the prediction of progression has been difficult, and the results of studies have been conflicting. Henoch-Schönlein nephritis (HSN) is rare in adults, and prediction of the outcome is even more difficult than in IgAN. This study was conducted to evaluate the clinical and histopathological features and predictors of the outcome of IgAN and HSN diagnosed in one centre (313 IgAN patients and 38 HSN patients), and especially in patients with normal renal function at the time of renal biopsy. The study also aimed to evaluate whether there is a difference in the progression rates in four countries (259 patients from Finland, 112 from UK, 121 from Australia and 274 from Canada), and if so, can this be explained by differences in renal biopsy policy. The third aim was to measure urinary excretions of cytokines interleukin 1ß (IL-1ß) and interleukin 1 receptor antagonist (IL-1ra) in patients with IgAN and HSN and the correlations of excretion of these substances with histopathological damage and clinical factors. A large proportion of the patients diagnosed in Helsinki as having IgAN had normal renal function (161/313 patients). Four factors, (hypertension, higher amounts of urinary erythrocytes, severe arteriolosclerosis and a higher glomerular score) which independently predicted progression (logistic regression analysis), were identified in mild disease. There was geographic variability in renal survival in patients with IgAN. When age, levels of renal function, proteinuria and blood pressure were taken into account, it showed that the variability related mostly to lead-time bias and renal biopsy indications. Amount of proteinuria more than 0.4g/24h was the only factor that was significantly related to the progression of HSN. the Hypertension and the level of renal function were found to be factors predicting outcome in patients with normal renal function at the time of diagnosis. In IgAN patients, IL-1ra excretion into urine was found to be decreased as compared with HSN patients and healthy controls. Patients with a high IL-1ra/IL-1ß ratio had milder histopathological changes in renal biopsy than patients with a low/normal IL-1ra/IL-1ß ratio. It was also found that the excretion of IL-1ß and especially IL-1ra were significantly higher in women. In conclusion, it was shown that factors associated with outcome can reliably be identified even in mild cases of IgAN. Predicting outcome in adult HSN, however, remains difficult.

Pathophysiology of Congenital Nephrotic Syndrome of the Finnish type

Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal recessive disease which is highly enriched in the Finnish population. It is caused by mutations in the NPHS1 gene encoding for nephrin, which is a major component of the glomerular filtration barrier in the kidney. Patients with NPHS1 have heavy proteinuria and nephrotic syndrome (NS) from birth and develop renal fibrosis in early childhood. Renal transplantation (TX) is the only curative treatment for NPHS1. These patients form the largest group of pediatric kidney transplant children in our country. The NPHS1 kidneys are removed in infancy and they serve as an excellent human material for studies of the pathophysiology of proteinuric kidney diseases. Sustained proteinuria is a major factor leading to end-stage renal failure and understanding this process is crucial for nephrology. In this study we investigated the glomerular and tubulointerstitial changes that occur in the NPHS1 kidneys during infancy as well as the expression of nephrin in non-renal tissues. We also studied the pathology and management of recurrent proteinuria in kidney grafts transplanted to NPHS1 children. Severe renal lesions evolved in patients with NPHS1 during the first months of life. Glomerular sclerosis developed through progressive mesangial sclerosis, and capillary obliteration was an early consequence of this process. Shrinkage of the glomerular tuft was common, whereas occlusion of tubular opening or protrusion of the glomerular tuft into subepithelial space or through the Bowman's capsule were not detected. Few inflammatory cells were detected in the mesangial area. The glomerular epithelial cells (podocytes) showed severe ultrastructural changes and hypertrophy. Podocyte proliferation and apoptosis were rare, but moderate amounts of podocytes were detached and ended up in the urine. The results showed that endocapillary lesions not extracapillary lesions, as generally believed were important for the sclerotic process in the NPHS1 glomeruli. In the tubulointerstitium, severe lesions developed in NPHS1 kidneys during infancy. Despite heavy proteinuria, tubular epithelial cells (TECs) did not show transition into myofibroblasts. The most abundant chemokines in NPHS1 tissue were neutrophil activating protein-2 (NAP-2), macrophage inhibiting factor (MIF), and monocyte chemoattractant protein-1 (MCP-1). Interstitial inflammation and fibrosis were first detected in the paraglomerular areas and the most abundant inflammatory cells were monocytes/macrophages. Arteries and arterioles showed intimal hypertrophy, but the pericapillary microvasculature remained quite normal. However, excessive oxidative stress was evident in NPHS1 kidneys. The results indicated that TECs were relatively resistant to the heavy tubular protein load. Nephrin was at first thought to be podocyte specific, but some studies especially in experimental animals have suggested that nephrin might also be expressed in non-renal tissues such as pancreas and central nervous system. The knowledge of nephrin biology is important for the evaluation of nephrin related diseases. In our study, no significant amounts of nephrin protein or mRNA were detected in non-renal tissues of man and pig as studied by immunohistochemistry and in situ hybridization. The phenotype analysis of NPHS1 children, who totally lack nephrin, revealed no marked impairment in the neurological, testicular, or pancreatic function speaking against the idea that nephrin would play an important functional role outside the kidney. The NPHS1 kidneys do not express nephrin and antibodies against this major glomerular filter protein have been observed in NPHS1 children after renal TX most likely as an immune reaction against a novel antigen. These antibodies have been associated with the development of recurrent NS in the kidney graft of NPHS1 patients. In our study, a third of the NPHS1 patients homozygous for Fin-Major mutation developed recurrent NS in the transplanted graft. Re-transplantations were performed to patients who lost their graft due to recurrent NS and heavy proteinuria immediately developed in all cases. While 73% of the patients had detectable serum anti-nephrin antibodies, the kidney biopsy findings were minimal. Introduction of plasma exchange (PE) to the treatment of recurrent nephroses increased the remission rate from 54% to 89%. If remission was achieved, recurrent NS did not significantly deteriorate the long term graft function. In conclusion, the results show that the lack of nephrin in podocyte slit diaphragm in NPHS1 kidneys induces progressive mesangial expansion and glomerular capillary obliteration and inflicts interstitial fibrosis, inflammation, and oxidative stress with surprisingly little involvement of the TECs in this process. Nephrin appears to have no clinical significance outside the kidney. Development of antibodies against nephrin seems to be a major cause of recurrent NS in kidney grafts of NPHS1 patients and combined use of PE and cyclophosphamide markedly improved remission rates.