Immo Rantala

Androgen Receptor Alterations in Prostate Cancer Relapsed during a Combined Androgen Blockade by Orchiectomy and Bicalutamide

Mechanisms of prostate cancer (CaP) recurrence during a combined androgen blockade (CAB) are poorly understood. Previously, the role of androgen receptor (AR) gene mutations underlying the CAB therapy relapse has been raised. To investigate the hypothesis that AR gene aberrations are involved in CAB relapse, 11 locally recurrent CaP samples from patients treated with orchiectomy and bicalutamide were analyzed for copy number changes and DNA sequence alterations of the AR gene by fluorescence in situ hybridization and single-strand conformation polymorphism, respectively. Altogether, base changes were detected in four tumors (36%). Three of them were missense mutations (G166S, W741C, M749I) and two were silent polymorphisms. Interestingly, none of the tumors had AR amplification. These data suggest that different AR variants are developed and selected for during various types of hormonal treatments, and also, that CAB achieved by orchiectomy and bicalutamide does not act as a selective force for AR amplification.

The effect of topical oestradiol on skin collagen of postmenopausal women

Objective To examine the effect of topical oestradiol on skin collagen and elastin.

Diet during rotavirus enteritis affects jejunal permeability to macromolecules in suckling rats

ABSTRACT: We studied the influence of diet during diarrhea on gut mucosal barrier in a suckling rat model. Rat pups were inoculated with IDIR virus (a group B rotavirus) at 10 d of age. Beginning 2 d postinfection, in addition to maternal milk, group CM received a daily gavage of cow milk and group GG received Lactobacillus casei strain GG, a human strain previously shown to survive the passage through the gastrointestinal tract and temporarily colonize the gut. Group CMGG received a combination of these, and control animals were gavaged with tap water. At 21 d of age, jejunal absorption of intact and degraded horseradish peroxidase (HRP) in Ussing chamber was markedly higher in IDIR virus-infected than in noninfected controls. In the two groups gavaged with cow milk, group CM and group CMGG, the numbers of specific antibody-secreting cells (enumerated by the solid-phase enzyme-linked immunospot assay) against β-lactoglobulin were significantly higher than in the groups that had not received cow milk. In parallel with immune system activation, a statistically significant increase in the absorption of intact HRP (mean and 95% confidence interval, ng × h−1 × cm−2) was detected: group CM, 302 (155, 586); group CMGG, 174 (56, 545); infected controls, 121 (57, 257); and group GG, 44 (8, 254). A decrease in the uptake of intact HRP (F = 3.64, p = 0.06) and degraded HRP (F = 9.50, p = 0.004) was associated with the introduction of L. casei GG to the diet, irrespective of coexposure to cow milk. These results indicate that feeding cow milk amplifies and intestinal implantation of lactobacilli may counteract rotavirus infection-associated intestinal dysfunction. They further suggest that milk fermented with lactic acid bacteria that are able to colonize the gut may prove near-optimal in dietary management of acute gastroenteritis.

Type 1 Diabetes Is Associated With Enterovirus Infection in Gut Mucosa

Enterovirus infections have been linked to type 1 diabetes in several studies. Enteroviruses also have tropism to pancreatic islets and can cause β-cell damage in experimental models. Viral persistence has been suspected to be an important pathogenetic factor. This study evaluates whether gut mucosa is a reservoir for enterovirus persistence in type 1 diabetic patients. Small-bowel mucosal biopsy samples from 39 type 1 diabetic patients, 41 control subjects, and 40 celiac disease patients were analyzed for the presence of enterovirus using in situ hybridization (ISH), RT-PCR, and immunohistochemistry. The presence of virus was compared with inflammatory markers such as infiltrating T cells, HLA-DR expression, and transglutaminase 2–targeted IgA deposits. Enterovirus RNA was found in diabetic patients more frequently than in control subjects and was associated with a clear inflammation response in the gut mucosa. Viral RNA was often detected in the absence of viral protein, suggesting defective replication of the virus. Patients remained virus positive in follow-up samples taken after 12 months’ observation. The results suggest that a large proportion of type 1 diabetic patients have prolonged/persistent enterovirus infection associated with an inflammation process in gut mucosa. This finding opens new opportunities for studying the viral etiology of type 1 diabetes.

Pattern of somatic androgen receptor gene mutations in patients with hormone-refractory prostate cancer.

Progression to hormone-refractory growth of prostate cancer has been suggested to be mediated by androgen receptor (AR) gene alterations. We analyzed AR for mutations and amplifications in 21 locally recurrent prostate carcinomas treated with orchiectomy, estrogens, or a combination of orchiectomy and estramustine phosphate using fluorescence in situ hybridization, single-strand conformation polymorphism, and DNA sequence analyses. Amplification was observed in 4 of 16 (25%) and amino acid changing mutations was observed in 7 of 21 (33%) of the tumors, respectively. Two (50%) tumors with AR amplification also had missense mutation of the gene. Four of five (80%) cancers that were treated with a combination of orchiectomy and estramustine phosphate had a mutation clustered at codons 514 to 533 in the N-terminal domain of AR. In functional studies, these mutations did not render AR more sensitive to testosterone, dihydrotestosterone, androstenedione, or β-estradiol. Tumors treated by orchiectomy had mutations predominantly in the ligand-binding domain. In summary, we found molecular alterations of AR in more than half of the prostate carcinomas that recurred locally. Some tumors developed both aberrations, possibly enhancing the cancer cell to respond efficiently to low levels of androgens. Furthermore, localization of point mutations in AR seems to be influenced by the type of treatment.

A quantitative method for the assessment of intraepidermal nerve fibers in small–fiber neuropathy

AbstractObjectives The purpose of this paper is to present an easy–to–use and reproducible morphometrical method of determining the density of intraepidermal nerve fibers (IENF) per epidermal area with the corresponding reference range of the IENF–counts.Methods Thirty patients and 22 controls were included in this study. The patients were divided into three groups: small–fiber (SFN), diabetic and demyelinating neuropathy. All subjects underwent punch skin biopsy. Specimens were fixed routinely in formalin and thereafter embedded in paraffin. Nerve fibers were revealed using immunoperoxidase staining with panaxonal antibody PGP 9.5. Using light microscopy, immunopositive nerves were counted morphometrically per epidermal area (NPEA) and, for comparison, per epidermal length (NPEL).Results Both the NPEA and NPEL estimates of SFN and diabetic neuropathy group differed significantly from those of control specimen (p < 0.001 and p < 0.001, Mann–Whitney test). Our method of counting, NPEA, shows a good correlation to NPEL (r = 0.945).Conclusions IENF–counting by a new morphometric modification is reproducible and diagnostically sensitive and can easily be adopted in any laboratory familiar with the basic immunohistochemical methodology. The method is less dependent on costly technical support systems and seems to be less time consuming when compared with conventional methods for IENF–counting.

Fecal Calprotectin Levels and Serological Responses to Microbial Antigens Among Children and Adolescents with Inflammatory Bowel Disease

Background: Noninvasive, sensitive, and specific tools for early identification of chronic inflammatory bowel disease (IBD) are needed for clinical practice. The aim was to identify new noninvasive test combinations for characterization of IBD in children and adolescents by comparing serological responses to microbial antigens and fecal calprotectin, a new promising marker for intestinal inflammation. Methods: Our study included 73 children who underwent endoscopies because of suspicion of IBD. Their sera were tested for antibodies to the Pseudomonas fluorescens‐associated sequence I2, a Bacteroides caccae TonB‐linked outer membrane protein, OmpW, and anti‐Saccharomyces cerevisiae (ASCA). Simultaneously, samples for fecal calprotectin measurements were obtained from 55 subjects. Results: IBD was diagnosed in 60 patients (Crohns disease [CD] in 18 patients, ulcerative colitis [UC] in 36, and indeterminate colitis [IC] in 6). Thirteen children had a non‐IBD disease. Fecal calprotectin levels were elevated (≥100 &mgr;g/g) more frequently in IBD patients (89%, 39/44) compared to non‐IBD cases (9%, 1/11, P < 0.001). ASCA antibodies in sera were detected in 67% (12/18) of patients with CD, in 14% (5/36) of the children with UC, and in 50% (3/6) of patients with IC. Seroreactivity for I2 was observed in 42% of the IBD patients, this frequency being higher than in non‐IBD cases (7.7% seropositive; P = 0.025). Serum anti‐I2 IgA levels (median absorbances) were higher in those with IBD compared to those without gut inflammation (P = 0.039). The combination of the measurements of fecal calprotectin and serological responses to microbial antigens (ASCA, I2, and OmpW) identified 100% of CD patients (sensitivity 100%, specificity 36%, positive predictive value [PPV] 66%, negative predictive value [NPV] 100%) and 89% of UC patients (sensitivity 89%, specificity 36%, PPV 77%, NPV 57%). Conclusions: Increased levels of serological responses to microbial antigens (ASCA, I2, and OmpW) and fecal calprotectin are evident in both CD and UC patients. The combination of these markers provides valuable, noninvasive tools for the diagnosis of IBD.

Identification of an alternatively spliced isoform of carbonic anhydrase XII in diffusely infiltrating astrocytic gliomas

Carbonic anhydrase XII (CA XII) is a transmembrane enzyme that is associated with neoplastic growth. CA XII has been proposed to be involved in acidification of the extracellular milieu, creating an appropriate microenvironment for rapid tumor growth. Because RNA sequence databases have indicated that two isoforms of CA XII might exist in human tissues, and because alternatively spliced protein forms have been linked to aggressive behavior of cancer cells, we designed a study to evaluate the presence of the two forms of CA XII in diffuse astrocytomas, a tumor type known for its aggressive and often noncurable behavior. Reverse transcription PCR of tumor samples surprisingly revealed that CA XII present in diffuse astrocytomas is mainly encoded by a shorter mRNA variant. We further showed by Western blotting that anti-CA XII antibody recognized both isoforms in the glioblastoma cell lines, and we then evaluated the expression of CA XII in astrocytomas using immunohistochemistry and correlated the results with various clinicopathological and molecular factors. Of 370 diffusely infiltrating astrocytomas, 363 cases (98%) showed immunoreactions for CA XII. Importantly, CA XII expression correlated with poorer patient prognosis in univariate (p = 0.010, log-rank test) and multivariate survival analyses (p = 0.039, Cox analysis). From these results, we conclude that CA XII is commonly expressed in diffuse astrocytomas and that it might be used as a biomarker of poor prognosis. The absence of 11 amino acids in the shorter isoform, which seems to be common in astrocytomas, may affect the normal quaternary structure and biological function of CA XII.

Proliferative Activity of Human Uterine Leiomyoma Cells as Measured by Automatic Image Analysis

Proliferative activity of uterine leiomyomas from premenopausal (n = 44) and postmenopausal (n = 12) women was investigated by automatic image analysis on frozen tissue sections using immunohistochemistry with anti-proliferating cell nuclear antigen antibody. The quantitative proliferation index (QPI) in premenopausal leiomyoma cells was significantly (p < 0.025) higher than that in leiomyomas in postmenopausal women. Leiomyomas proliferated most actively during the secretory phase. After the climacterium, leiomyomas showed no proliferative activity in the absence of hormone supply, while combined substitution with estrogen and progestagen considerably increased QPI.

Amplification of the androgen receptor gene is associated with P53 mutation in hormone-refractory recurrent prostate cancer.

p53 protein expression of 30 hormone‐refractory locally recurrent prostate cancers was compared with their matched untreated primary tumour specimens. In addition, androgen receptor (AR) gene amplification and p53 protein immunostaining were compared. p53 positivity increased during hormonal therapy from 17 per cent of the untreated primary tumours to 40 per cent of the hormone‐refractory recurrences (p = 0·078). None of the p53‐positive primary tumour specimens lost p53 positivity during hormonal therapy. Hormone‐refractory recurrences with AR gene amplification more frequently (p = 0·0342) showed positive p53 immunostaining than tumours without AR gene amplification, 75 and 27 per cent, respectively. In summary, this study has shown that a cell clone with P53 mutation seems to be selected for during endocrine therapy and that positive p53 immunostaining correlates with AR gene amplification. These results suggest that inactivation of P53 may lead to genetic instability in a subset of prostate carcinomas enabling them to achieve properties, such as AR gene amplification, that allow them to grow in low levels of androgens and therefore cause tumour progression. Copyright