Number of nexin links detectable at standard electron microscopy of normal human nasal cilia and at nexin link deficiency.

BACKGROUND: Eleven years ago we had described three patients with missing nexin links as a possible cause of primary ciliary dyskinesia (PCD). The assumption was substantiated last year by finding a mutation in these patients. MATERIALS AND METHODS: We counted the nexin links, inner (IDA) and outer (ODA) dynein arms and microtubuli in each of, if possible, 50 cilia in 41 patients with normal cilia, 4 patients with deficiency of nexin links only and 4 with deficiency of nexin links and IDA. RESULTS: In the control group the median number of nexin links was 4.5 per cilium, range 3.4-5.3. In the second group the mean numbers of nexin links per cilium were 1.1-1.4, in the third group 0.8-1.2, per patient. The median number of IDA was in the control group 4.2, range 3.3-5.2. In groups 2 and 3 the numbers were 3.0-3.5 and 0.2-1.0, respectively. Numbers of ODA were normal in all groups. CONCLUSIONS: It is possible to reliable count the number of nexin links in nasal human cilia and to distinguish cases with missing nexin links from normal controls.

Changes in immunocompetent cells after interstitial laser thermotherapy of breast cancer.

BACKGROUND: Local tumour destruction has been shown to give rise to changes in immunocompetent cells. The aim of this study was to describe the effect of interstitial laser thermotherapy (ILT) of breast carcinoma in the tumour and in regional lymph nodes. METHODS: Seventeen women that underwent radical surgical excision after non-radical ILT were studied. ILT was performed at a steady-state temperature of 48°C for 30 min. Surgical excision was performed 12 (6-23) days after ILT. Six patients with breast cancer not treated with ILT before surgery served as controls. Immunohistological reactions were performed on core needle biopsies prior to treatment and on the excised specimens. RESULTS: ILT resulted in more CD8 lymphocytes and CD68 macrophages within the tumour (P < 0.05 and P < 0.01, respectively) and higher counts of CD20 (P < 0.05), CD68 (P < 0.001) and CD83 (P < 0.01) at the tumour border, when compared to pre-treatment values. In the control patients not receiving ILT, CD8 cells increased within the tumour after resection (P < 0.05). With the probable exception of CD25 Foxp3 cells, the presence of cancer in a lymph node influenced the findings in lymph nodes (examined for CD1a, CD25, Foxp3 CD25, CD83 cells). Thus, comparisons between ILT and control patients were restricted to patients without lymph node metastases. In these patients, ILT and resection were followed by a decrease in CD25 Foxp3 lymphocytes (P < 0.05), when compared to surgical resection alone. CONCLUSIONS: ILT induced changes in immunocompetent cells in patients with breast cancer. The stimulation of the immune system is an added feature of ILT in treatment of patients with breast cancer.

Intimal hyperplasia in balloon dilated coronary arteries is reduced by local delivery of the NO donor, SIN-1 via a cGMP-dependent pathway.

BACKGROUND: To elucidate the mechanism by which local delivery of 3-morpholino-sydnonimine (SIN-1) affects intimal hyperplasia after percutaneous transluminal coronary angioplasty (PTCA). METHODS: Porcine coronary arteries were treated with PTCA and immediately afterwards locally treated for 5 minutes, with a selective cytosolic guanylate cyclase inhibitor, 1 H-(1,2,4)oxadiazole(4,3-alpha)quinoxaline-1-one (ODQ) + SIN-1 or only SIN-1 using a drug delivery-balloon. Arteries were angiographically depicted, morphologically evaluated and analyzed after one and eight weeks for actin, myosin and intermediate filaments (IF) and nitric oxide synthase (NOS) contents. RESULTS: Luminal diameter after PCI in arteries treated with SIN-1 alone and corrected for age-growth was significantly larger as compared to ODQ + SIN-1 or to controls (p < 0.01). IF/actin ratio after one week in SIN-1 treated segments was not different compared to untreated segments, but was significantly reduced compared to ODQ + SIN-1 treated vessels (p < 0.05). Expression of endothelial NADPH diaphorase activity was significantly lower in untreated segments and in SIN-1 treated segments compared to controls and SIN-1 + ODQ treated arteries (p < 0.01). Restenosis index (p < 0.01) and intimal hyperplasia (p < 0.01) were significantly reduced while the residual lumen was increased (p < 0.01) in SIN-1 segments compared to controls and ODQ + SIN-1 treated vessels. CONCLUSIONS: After PTCA local delivery of high concentrations of the NO donor SIN-1 for 5 minutes inhibited injury induced neointimal hyperplasia. This favorable effect was abolished by inhibition of guanylyl cyclase indicating mediation of a cyclic guanosine 3',5'-monophosphate (cGMP)-dependent pathway. The momentary events at the time of injury play crucial role in the ensuring development of intimal hyperplasia.

Interstitial laser thermotherapy of a rat liver tumour: effect of hepatic inflow occlusion.

BACKGROUND AND OBJECTIVE: Interstitial laser thermotherapy was used to treat rat liver tumours. The aim was to investigate the influence of temperature and temporary hepatic inflow occlusion on tumour growth and blood perfusion. STUDY DESIGN/MATERIALS AND METHODS: Liver tumours were treated at 44°C at the tumour border for 30 minutes, hepatic inflow occlusion only, or a combination of these methods. Interstitial laser Doppler flowmetry was used to measure hepatic perfusion at the tumour border during and after heat treatment, for a total time of 60 minutes. Tumour growth was evaluated 6 days after treatment. RESULTS: Tumours subjected to the combined treatment of hepatic inflow occlusion and interstitial laser thermotherapy displayed a blood perfusion reduction 30 minutes after treatment to 18 ± 5% of initial perfusion, which was significantly lower than achieved with thermotherapy alone (52 ± 10%, P = 0.02). The combined treatment and treatment with thermotherapy alone resulted in relative tumour growth of 0.3 ± 0.1 and 1.0 ± 0.2, respectively (P = 0.04). CONCLUSION: Inflow occlusion enhanced the effect of thermotherapy not by augmenting treatment temperatures but by increasing the thermal sensitivity of the tumour, reflected by an immediate effect on tumour blood perfusion.

Perivascular epithelioid cell tumour of the liver.

Perivascular epithelioid cell tumour is not uncommon in the liver but seldom malignant.

First attempt to produce experimental Campylobacter concisus infection in mice.

AIM: To infect mice with atypical Campylobacter concisus (C. concisus) for the first time. METHODS: Three separate experiments were conducted in order to screen the ability of five clinical C. concisus isolates of intestinal origin and the ATCC 33237 type strain of oral origin to colonize and produce infection in immunocompetent BALB/cA mice. The majority of the BALB/cA mice were treated with cyclophosphamide prior to C. concisus inoculation to suppress immune functions. Inoculation of C. concisus was performed by the gastric route. RESULTS: C. concisus was isolated from the liver, ileum and jejunum of cyclophosphamide-treated mice in the first experiment. No C. concisus strains were isolated in the two subsequent experiments. Mice infected with C. concisus showed a significant loss of body weight from day two through to day five of infection but this decreased at the end of the first week. Histopathological examination did not consistently find signs of inflammation in the gut, but occasionally microabscesses were found in the liver of infected animals. CONCLUSION: Transient colonization with C. concisus was observed in mice with loss of body weight. Future studies should concentrate on the first few days after inoculation and in other strains of mice.

Prevalence and genetics of immotile-cilia syndrome and left-handedness.

Immotile-cilia syndrome is characterized by severe respiratory distress from early infancy, and also often by situs inversus. The first description of the disease was based on just four persons, but reasons were given to suggest that the disorder may not be exceedingly rare. The purpose of the present study was to estimate just how rare or how common it is and to evaluate its association with situs inversus and with left-handedness. Data were mainly obtained from contacting a large number of Swedish clinicians who kindly informed us about their patients with suspected immotile-cilia syndrome. Diagnosis was in most cases performed by electron microscopical examination of nasal cilia or of spermatozoa. Based on these data, the prevalence of the syndrome in Sweden with or without situs inversus was estimated to be not far from 1 in 10,000. The syndrome consists of several subgroups that have a randomized determination of situs asymmetry (50% of these have situs inversus) and one subgroup in which situs inversus is not found. This results in a frequency of situs inversus in the syndrome of about 44 %. Left-handedness is no more common than it is in healthy persons and no more often associated with situs inversus than with situs solitus. In all cases it is about 14 %. It is concluded that the two major anatomical/physiological asymmetries of the human body are found with frequencies which indicate that they develop independently of each other. Both conditions appear with prevalences that may have changed at a centenary scale, left-handedness with a substantial increase and situs inversus with a less dramatic increase.

Helicobacter species ribosomal DNA in the pancreas, stomach and duodenum of pancreatic cancer patients.

AIM: To determine whether gastric and enteric Helicobacter species are associated with pancreatic cancer. METHODS: Patients with exocrine pancreatic cancer (n = 40), neuroendocrine cancer (n = 14), multiple endocrine neoplasia type 1 (n = 8), and chronic pancreatitis (n = 5) were studied. Other benign pancreatic diseases (n = 10) and specimens of normal pancreas (n = 7) were included as controls. Pancreatic tissue specimens were analyzed by Helicobacter-specific PCR-assay and products were characterized by denaturing gradient electrophoresis and DNA-sequencing. From a subset of the pancreatic cancer patients, gastric and/or duodenal tissue as well as gallbladder and ductus choledochus tissue were analyzed. Gallbladder and choledochus samples were included as controls. Stomach and duodenum samples were investigated to analyze whether a gastric helicobacter might disseminate to the pancreas in pancreatic cancer patients. Pancreatic specimens were analyzed by Bacteroides-specific PCR for detecting the translocation of indigenous gut microbes to the diseased pancreas. RESULTS: Helicobacter DNA was detected in pancreas (tumor and/or surrounding tissue) of 75% of patients with exocrine cancer, 57% of patients with neuroendocrine cancer, 38% of patients with multiple endocrine neoplasia, and 60% of patients with chronic pancreatitis. All samples from other benign pancreatic diseases and normal pancreas were negative. Thirty-three percent of the patients were helicobacter-positive in gastroduodenal specimens. Surprisingly, H. bilis was identified in 60% of the positive gastroduodenal samples. All gallbladder and ductus choledochus specimens were negative for helicobacter. Bacteroides PCR-assay was negative for all pancreatic samples. CONCLUSION: Helicobacter DNA commonly detected in pancreatic cancer suggests a possible role of the emerging pathogens in the development of chronic pancreatitis and pancreatic cancer.

Photodynamic therapy of nodular basal cell carcinoma with multifiber contact light delivery.

To overcome the limited treatment depth of superficial photodynamic therapy we investigate interstitial light delivery. In the present work the treatment light was delivered using a system in which three or six clear-cut fibers were placed in direct contact with the tumor area. This placement was thought to represent a step toward general purpose interstitial PDT. Twelve nodular basal cell carcinomas were treated employing delta-aminolevulinic acid and 635 nm laser irradiation. Fluorescence measurements were performed monitoring the buildup and subsequent bleaching of the produced sensitizer protoporphyrin IX. The treatment efficacy, judged at a 28-month follow-up, showed a 100% complete response. Two punch excisions at 7 months converted two partial responses to complete responses. One patient failed to appear at all follow-up sessions. The outcome of the treatments was comparable to superficial photodynamic therapy in terms of histological, clinical, and cosmetic results.

Methods for detailed histopathological investigation and localization of biopsies from cervix uteri to improve the interpretation of autofluorescence data.

Fluorescence spectroscopy is one of many optical methods that are potentially clinically useful for noninvasive detection and characterization of disorders on the cervical part of uterus, including precancerous lesions. The cervix uteri exhibits a biologically complex tissue and the morphology of a biopsy is generally not homogenous. The standard histopathological protocol accounts only for the most severe condition found within the biopsy and no information is given on other constituents potentially influencing the recorded fluorescence spectra. Spectra are usually correlated, using multivariate techniques, to the histopathological diagnosis of the biopsies. Since the probe volume of fluorescence spectroscopy is considerably smaller than the extension of the biopsy, this can cause problems in the search for correlation between the fluorescence signals and the pathological structures. In addition, the orientation and location of the biopsies are normally not recorded. We now report on the first detailed histopathological protocol where numerous tissue parameters, such as thickness and type of the epithelium and the number of blood vessels, glands, and inflammatory cells, are tabulated and the orientation and location of the biopsy are recorded as precisely as possible. Hopefully, the use of this protocol together with sophisticated mathematical methods will increase the probability to classify cervical disorders of the uterus, including precancerous lesions, with high sensitivity and specificity.

Long-term Follow-up After Interstitial Laser Thermotherapy of Breast Cancer.

AIM: To review the effect of immunological changes induced by interstitial laser thermotherapy (ILT) on long-term outcome of patients with breast cancer. PATIENTS AND METHODS: Twenty-four patients with invasive breast cancer were treated with ILT followed by standard surgical excision. Immunohistological reactions on immunocompetent cells were performed on specimens obtained before and after ILT. Follow-up time was 116 (range=91-136) months. RESULTS: Significant prognostic factors were histologically-positive axillary lymph nodes and Ki67 positivity. ILT increased cytotoxic T (CD8(+)) lymphocytes within the tumor and mature dendritic cells (CD83(+)) and reduced the number of T-regulatory cells (Treg) CD25(+)/Forkhead box p3(+) (FOXP3(+)) lymphocytes in regional lymph nodes. These changes did not correlate with prognosis. The number of CD8(+) cells within the tumor, both before and after treatment, was significantly higher in patients with recurrence than in those without recurrence (p<0.01 and p<0.05, respectively). Patients with recurrent disease had a lower number of CD57(+) cells in tumor-free lymph nodes than did patients without recurrence (p<0.05). CONCLUSION: ILT did not have any long-term adverse effects. The clinical impact of the supposedly favourable immune changes after ILT should be examined in a larger patient population.

Linomide improves the effect of interstitial laser thermotherapy in a rat liver tumour model.

AIM: The aim of this study was to investigate if Linomide affects growth and spread of a rat liver tumour when given alone and in combination with interstitial laser thermotherapy (ILT). MATERIALS AND METHODS: Experiments were performed in Wistar rats using a dimethyl-hydrazine-induced adenocarcinoma implanted into the left lateral lobe of the liver. The rats were randomised to one of the following groups: a) ILT and Linomide, b) ILT only, c) sham ILT, d) Linomide only, or e) control. ILT was intentionally suboptimal. Linomide (100 mg/kg/day) was given in the drinking water from the start of treatment for five days. ED1, ED2 macrophages and v Willebrand (factor VIII) were determined by an immunohistochemical technique. RESULTS: Linomide reduced viable liver tumour volume both when it was given alone (p < 0.01) and when combined with ILT (p < 0.05), whereas it lowered intraperitoneal spread in ILT-treated rats alone. Six days after ILT, there was a reduction in the number of newly-recruited macrophages and blood vessels in the viable tumour tissue in rats receiving Linomide. CONCLUSION: Linomide reduced the growth of an adenocarcinoma transplanted into rat liver, when given alone or combined with laser thermotherapy and reduced the spread of tumour in laser-treated rats. The effects of Linomide in laser-treated rats appeared, at least in part, to be due to a reduction in newly-formed vessels, which might have been secondary to a reduced number of tumour-associated macrophages.

Heat shock protein 70 (HSP70) after laser thermotherapy of an adenocarcinoma transplanted into rat liver.

The heat shock proteins (HSPs) HSP70 and gp96 from necrotic tumour cells are considered to function as chaperones in presenting tumour antigens. We therefore studied HSP70 and immune cells in a transplantable carcinoma in the liver of rats after interstitial laser thermotherapy (ILT). Experiments were performed in Wistar FU rats using a dimethyl-hydrazine-induced adenocarcinoma implanted into the left lateral lobe of the liver. Rats were randomized to one of the following groups: a) ILT of tumour, b) sham ILT, or c) control. ILT was suboptimal and was performed at a steady-state temperature of 43 degrees C at the tumour margin for 30 minutes. Rats were killed 15 minutes, 5 hours, 10 hours, 15 hours or 12 days after treatment. Double immunohistochemistry was performed for HSP70 and ED1 macrophages or CD8 lymphocytes, and ELISA for serum concentrations of HSP70. After ILT, there was an increase of HSP70 immunoreactivity in tumours as compared to sham ILT. At the same time, tumour cells affected by ILT showed a shift of HSP70 from the cytoplasm to the nucleus with a peak at 10 hours. Few CD8-positive cells were found. There was an increase of tumour-infiltrating ED1 macrophages after ILT as compared to sham ILT at 10-15 hours after treatment. HSP70 was present in ED1 macrophages significantly more frequently after ILT than after sham ILT, and this was true both for HSP70 localized to the surface and the cytoplasm of the macrophage. There was a significant increase in serum HSP70 during the first 15 hours after ILT. In conclusion, laser thermotherapy resulted in increased HSP70 immunoreactivity within tumours and HSP70 shifts from cytoplasm to nucleus. Furthermore, it resulted in increased numbers of tumour-infiltrating macrophages and an increased presence of HSP70 in the membrane and cytoplasm of these macrophages.

Effects of inhibition of glycation and oxidative stress on the development of diabetic nephropathy in rats.

We investigated whether aminoguanidine (AG), an inhibitor of advanced glycated end product formation, or probucol (PB), a free radical scavenger, could influence signs of glomerular and distal tubular function and morphological changes in kidneys of male Wistar rats after 6 months of streptozocin (STZ)-induced diabetes. Diabetic rats had a higher kidney weight/body weight ratio (P<.001), but neither AG nor PB influenced the increased ratio. Diabetes caused an increased urinary albumin excretion (P<.05), which was normalized by AG, but further exaggerated by PB (P<.001). Diabetes also caused an increase in the urinary excretion of Tamm-Horsfall protein (P<.001). Both AG and PB attenuated this increase (P<.05 for both). A few glomeruli displayed focal thickening of varying degrees. Silver staining disclosed the glomerulopathy to be intercapillary glomerulosclerosis. Rats on PB-enriched diet displayed less pronounced changes than untreated rats (P<.01), while AG had no effect. The results suggest that oxidative stress could be involved in the development of diabetic nephropathy.

Extended operation with or without intraoperative (IORT) and external (EBRT) radiotherapy for gallbladder carcinoma.

BACKGROUND/AIMS: Gallbladder carcinoma is a rare disease with dismal prognosis. However, lately improved survival has been reported after extended operation including liver resection and lymphadenectomy in addition to cholecystectomy. The aim of this study was to evaluate such a surgical strategy with and without adjuvant intra- and postoperative radiotherapy (IORT/EBRT). METHODOLOGY: 20 patients underwent extended operation and the last 10 of them IORT/EBRT in addition. Tumor staging was done using the TNM system, determination of histological tumor differentiation and immunohistochemical assessment of p53, Ki67, metallothionein, deleted in colorectal cancer and carcinoembryogenic antigen in tumor tissue. RESULTS: There was no hospital mortality. Postoperative complications occurred in 3 patients (15%). Actuarial 5-year survival was 47% in the radiotherapy group and 13% after operation only (NS). The corresponding figures for median survival are 28.8 and 20.2 months, respectively. Five patients are still alive in the radiotherapy group. There was no difference in tumour stages of the two groups irrespective of the way of evaluation. CONCLUSIONS: The results suggest that extended operation for gallbladder carcinoma +/- IORT/EBRT can be done safely. The tendency to longer survival after adjuvant radiotherapy was not statistically significant.

The nexin-dynein regulatory complex subunit DRC1 is essential for motile cilia function in algae and humans.

Primary ciliary dyskinesia (PCD) is characterized by dysfunction of respiratory cilia and sperm flagella and random determination of visceral asymmetry. Here, we identify the DRC1 subunit of the nexin-dynein regulatory complex (N-DRC), an axonemal structure critical for the regulation of dynein motors, and show that mutations in the gene encoding DRC1, CCDC164, are involved in PCD pathogenesis. Loss-of-function mutations disrupting DRC1 result in severe defects in assembly of the N-DRC structure and defective ciliary movement in Chlamydomonas reinhardtii and humans. Our results highlight a role for N-DRC integrity in regulating ciliary beating and provide the first direct evidence that mutations in DRC genes cause human disease.

Helicobacter species and gut bacterial DNA in Meckel's diverticulum and the appendix.

AIM: To analyse the possible association of various Helicobacter species and certain common gut bacteria in patients with Meckel's diverticulum and appendicitis. METHODS: A nested-polymerase chain reaction (PCR), specific to 16S rRNA of the Helicobacter genus, was performed on paraffin embedded samples, 50 with acute appendicitis, 50 normal appendixes, and 33 Meckel's diverticulum with gastric heterotopia and/or ulcer. Helicobacter genus positive samples were sequenced for species identification. All samples were also analysed for certain gut bacteria by PCR. RESULTS: Helicobacter pullorum DNA was found in one out of 33 cases and Enterobacteria in two cases of Meckel's diverticulum. Helicobacter pylori (H. pylori) was found in three, Enterobacter in 18, and Bacteroides in 19 out of 100 appendix samples by PCR. Enterococcus was not found in any MD or appendix samples. All H. pylori positive cases were from normal appendixes. CONCLUSION: Helicobacter is not an etiological agent in the pathogenesis of symptomatic Meckel's diverticulum or in acute appendicitis.

Histology of adipose tissue inflammation in Dercum's disease, obesity and normal weight controls: a case control study.

BACKGROUND: Dercum's disease (DD) is characterised by obesity and chronic pain (> 3 months) in the adipose tissue. The pathogenesis of DD is unknown, but inflammatory components have been proposed. In previous reports and studies, an inconsistent picture of the histological appearance of the adipose tissue in DD has been described. The aim of this investigation was to examine the histological appearance of adipose tissue in patients with DD, with particular focus on inflammatory signs. METHODS: Fat biopsies were sampled from painful regions from 53 patients with DD. In 28 of the patients, a control adipose tissue biopsy was taken from a location where the patient did not experience any pain. In addition, fat biopsies were sampled from 41 healthy pain-free obese control patients and 11 healthy pain-free normal weight control patients. The extent of inflammation was evaluated on histological sections stained with haematoxylin-eosin. RESULTS: There was no statistically significant difference in the extent of inflammation between the biopsies from the painful knee and the biopsies from the non-painful area (p = 0.5), nor between the biopsies from the abdomen, and the biopsies from the non-painful area (p = 0.4), in patients with DD. A statistically significant difference in extent of inflammation was observed between DD and obese control patients regarding the abdomen (p = 0.022), but not the knee (p = 0.33). There were no differences in extent of inflammation between DD patients and normal weight controls (p = 0.81). CONCLUSION: The findings suggest that there is an inflammatory response in the adipose tissue in DD. However, this response is not more pronounced than that in healthy obese controls. This contradicts inflammation as the aetiology of DD.