Diagnostic value of immunohistochemistry on formalin-fixed, paraffin-embedded skin biopsy specimens for bullous pemphigoid.

BACKGROUND: Direct immunofluorescence microscopy (DIF) studies constitute the gold standard for diagnosis of bullous pemphigoid (BP) but depend on the availability of specialized laboratories and often on an additional skin biopsy specimen. OBJECTIVES: To assess the value of immunohistochemical analyses (IHCA) in the diagnosis of BP using formalin-fixed, paraffin-embedded skin biopsy specimens as an alternative to DIF; and to study the correlation between the results of IHCA and the presence of histological subepidermal blister formation and of circulating autoantibodies by indirect immunofluorescence studies using split skin or by enzyme-linked immunosorbent assays. METHODS: We included all patients newly diagnosed with BP evaluated between 2008 and 2010. There were 51 consecutive skin biopsy specimens obtained from 38 patients with BP with positive DIF. RESULTS: By IHCA, deposits of immunoreactants were found in 45% of all tested cases. Deposits of C3d, IgG, IgM, IgE and IgA were found in 37%, 23%, 2%, 0% and 0% of cases, respectively. Deposits of C3d and/or IgG were found in 79% of the 24 cases with a blister and in 83% of the 12 cases with subepidermal blistering and positive immunoserological analyses, respectively. CONCLUSIONS: In contrast to previous studies, our findings in an unselected patient cohort indicate that IHCA are not sufficiently sensitive to replace DIF studies for confirming the diagnosis of BP. IHCA sensitivity significantly increases in the presence of histological blistering and/or of circulating autoantibodies. IHCA represents a potential rescue diagnostic technique only if specialized laboratories and/or a second biopsy specimen for DIF are unavailable.

PrP expression in B lymphocytes is not required for prion neuroinvasion.

Prion diseases are typically initiated by infection of peripheral sites, as in the case of bovine spongiform encephalopathy, new variant Creutzfeldt-Jakob disease, kuru and most cases of iatrogenic Creutzfeldt-Jakob disease. In mouse scrapie, prion infectivity accumulates in lymphoid organs, and the absence of mature B lymphocytes prevents peripherally administered prions from inducing central nervous system disease. We have now assessed whether expression of the cellular prion protein, PrPc, is required for B lymphocytes to mediate neuroinvasion. We found that repopulation of SCID and Rag-1(-/-) mice with fetal liver cells from either PrP-expressing or PrP-deficient mice and from T-cell deficient mice, but not from B-cell deficient mice, is equally efficient in restoring neuroinvasion after intraperitoneal inoculation of scrapie prions. These results indicate that cells whose maturation depends on B cells or their products, such as follicular dendritic cells, may enhance neuroinvasion. Alternatively, B cells may transport prions to the nervous system by a PrP-independent mechanism.

Prion protein devoid of the octapeptide repeat region restores susceptibility to scrapie in PrP knockout mice.

Mice devoid of PrP are resistant to scrapie and fail to replicate the agent. Introduction of transgenes expressing PrP into such mice restores susceptibility to scrapie. We find that truncated PrP devoid of the five copper binding octarepeats still sustains scrapie infection; however, incubation times are longer and prion titers and protease-resistant PrP are about 30-fold lower than in wild-type mice. Surprisingly, brains of terminally ill animals show no histopathology typical for scrapie. However, in the spinal cord, infectivity, gliosis, and motor neuron loss are as in scrapie-infected wild-type controls. Thus, while the region comprising the octarepeats is not essential for mediating pathogenesis and prion replication, it modulates the extent of these events and of disease presentation.

Alteration of activator protein 1 DNA binding activity in gentamicin-induced hair cell degeneration.

Sensorineural hearing loss is often associated with damage of cochlear hair cells and/or of the neurons of the auditory pathway. This damage can result from a variety of causes, e.g. genetic disorders, aging, exposure to certain drugs such as aminoglycosides, infectious disease and intense sound overexposure. Intracellular events that mediate aspects of aminoglycoside-mediated damage to hair cells have been partially unraveled. Several independent research groups have demonstrated a crucial role of mitogen-activated protein kinase signaling in aminoglycoside-induced ototoxicity. Mitogen-activated protein kinases are important mediators of signal transduction from the cell surface to the nucleus. Jun N-terminal kinases, members of the mitogen-activated protein kinase family, are strongly activated in cell culture conditions by stress inducing stimuli, including ultraviolet light, heat shock and tumor necrosis factor; therefore they are also referred to as stress-activated protein kinases. In hair cells aminoglycoside treatment was shown to activate the Jun N-terminal kinase signaling pathway. Activation of Jun N-terminal kinase leads to phosphorylation and thereby activation of transcription factors and consequently to altered gene expression. There are many nuclear Jun N-terminal kinase substrates including c-Jun, ATF-2, and Elk-1 proteins. One of the downstream targets of Jun N-terminal kinase is the transcription factor activating protein-1. Activating protein-1 is a dimeric complex composed of members of the Fos and Jun proteins. A variety of different stimuli is known to induce activating protein-1 activity. Induction of activating protein-1 is thought to play a central role in reprogramming gene expression in response to external stimuli. In this study we have analyzed the effect of gentamicin treatment on the downstream targets of Jun N-terminal kinase. Our results demonstrate that gentamicin treatment of explants of organ of Corti results in increased activating protein-1 binding activity. The main component of these activating protein-1 complexes is the c-Fos protein. Moreover, we show that the activating protein-1 induction is transient and occurs exclusively in hair cells of rat organ of Corti explants.

Differential p53-dependent mechanism of radiosensitization in vitro and in vivo by the protein kinase C-specific inhibitor PKC412.

The cellular response to ionizing radiation is governed by the DNA-damage recognition process but is also modulated by cytoplasmic signal transduction cascades that are part of the cellular stress response. Growth-promoting protein kinase C activity antagonizes irradiation-induced cell death, and, therefore, protein kinase C inhibitors might be potent radiosensitizers. The antiproliferative and radiosensitizing effect of the novel N-benzoylated staurosporine analogue PKC412 was tested in vitro against genetically defined p53-wild type (+/+) and p53-deficient (-/-) murine fibrosarcoma cells and in vivo against radioresistant p53-/- murine fibrosarcoma and human colon adenocarcinoma tumor xenograft (SW480, p53-mutated). PKC412 sensitized both p53+/+ and p53-/- tumor cells in vitro and in vivo for treatment with ionizing radiation but with a different mechanism of radiosensitization depending on the p53 status. In p53+/+, cells combined treatment with PKC412 and ionizing radiation drastically induced apoptotic cell death, whereas no apoptosis induction could be observed in p53-deficient cells in vitro and in histological tumor sections. Combined treatment resulted in an increased G2 cell cycle distribution in p53-/- cells at PKC412 concentrations that did not alter cell cycle distribution when applied alone. In vivo, a minimal treatment regimen during 4 consecutive days of PKC412 (4 x 100 mg/kg) in combination with ionizing radiation (4 x 3 Gy) exerted a substantial tumor growth delay for both p53-disfunctional tumor xenografts and showed that the clinically relevant protein kinase C inhibitor PKC412 is a promising new radiosensitizer with a potentially broad therapeutic window.

Use of brain grafts to study the pathogenesis of prion diseases.

For the study of prion neurotoxicity, we used neural-grafting techniques: mice devoid of the normal host prion protein (Prnp% mice) received a neural graft and were intracerebrally infected with mouse prions. The growth and differentiation properties of neural grafts were defined. Growth of embryonic neuroectodermal tissue was optimal at gestational days 12.5-13.5. The blood-brain barrier is reconstituted after 7 weeks in most animals. Scrapie-infected PrPC-expressing grafts develop a severe spongiform encephalopathy and contain proteinase-resistant protein and infectivity. Infected grafts deliver high amounts of prions to the host brain without eliciting disease. Infected grafts show a progressive disruption of the blood-brain barrier. Following intraocular prion inoculation of a transplanted Prnp% mouse, prions do not reach the intracerebral graft, indicating that PrP expression is required for propagation along the optic tract.

An unusual case of Creutzfeldt-Jakob disease.

A patient with histopathologically verified sporadic Creutzfeldt-Jakob disease (CJD) presented initially with diplopia, sleep disturbances, and L-dopa-responsive parkinsonism. After more than a year of slow progression, he did not become demented, and failed to fulfill the clinical criteria for possible CJD. No clinical examinations currently proposed to detect CJD showed the disease. CJD should be in the differential diagnosis of "parkinson plus" syndromes until a different etiology has been found or a histopathologic examination performed.

Morphology of interstitial cells in the healthy kidney.

Renal interstitial cells play an important role in renal function and renal diseases. We describe the morphology of renal interstitial cells in the healthy kidney. We distinguish within the renal interstitium (1) renal fibroblasts and (2) cells of the immune system. Fibroblasts are in the majority and constitute the scaffold of the kidney; they are interconnected by junctions, and are attached to tubules and vessels. Although the phenotype of fibroblasts shows some variation depending on their location in the kidney and on their functional stage, their recognition as fibroblasts is possible on account of structural features. Among the cell types of the second group, antigen-presenting dendritic cells are the most abundant in in the peritubular interstitial spaces of healthy kidneys. Their incidence is highest in the inner stripe of the outer medulla. They share some morphological features with fibroblasts but lack others--junctional complexes, morphologically defined connections with tubules and vessels, and the prominent layer of actin filaments under the plasma membrane--that are characteristic for fibroblasts. Dendritic cells in healthy kidneys are morphologically different from macrophages, which are characterized by abundant primary and secondary lysosomes. In healthy kidneys macrophages are restricted to the connective tissue of the renal capsule and the pelvic wall, and to the periarterial connective tissue. Lymphocytes are rare in healthy kidneys. The distinction of cell types by morphology is supported by differences of membrane proteins. Among all interstitial cells in the renal cortex, fibroblasts alone exhibit ecto-5'-nucleotidase. Dendritic cells constitutively have a high abundance of MHC class II protein. Both proteins are mutually exclusive. Rat macrophages display the membrane antigen ED 2 and lymphocytes exhibit specific surface antigens, depending on their type and functional stage, e.g., CD4 or CD8.

Postnatal maturation of renal cortical peritubular fibroblasts in the rat.

The stromal cells in the renal cortex and medulla of adult rats reveal different phenotypes. Cortical peritubular fibroblasts are ecto-5'nucleotidase (5'NT)-positive and lack alpha-smooth muscle actin (alphaSMA) and vimentin immunoreactivity, whereas medullary fibroblasts are 5'NT-negative and vimentin-positive. We have studied by immunohistochemistry the postnatal (neonatal up to 8 weeks) development of renal cortical stromal cells with respect to 5'NT and to the cytoskeletal proteins alphaSMA and vimentin. Both alphaSMA and vimentin are characteristic for the renal myofibroblasts that replace stromal fibroblasts in interstitial nephritis. In new-born and 1-week-old rats, stromal cells in the cortex and medulla display alphaSMA and vimentin, but lack 5'NT. During the second postnatal week, alphaSMA and vimentin immunoreactivity in cortical interstitial cells gradually declines, whereas 5'NT reactivity becomes progressively apparent between the convoluted tubules in the juxtamedullary labyrinth. For a short time, all three proteins are found to be coexpressed in the same cells. At the end of the third week, interstitial 5'NT-immunoreactivity becomes evident also in the superficial cortical labyrinth, and alphaSMA and vimentin are no longer detectable in cortical peritubular cells. From the fourth week on, the distribution pattern and phenotype of 5'NT-positive cortical fibroblasts correspond to that in adult rats. The temporal pattern of maturation of cortical peritubular fibroblasts seems to parallel the functional maturation of cortical tubules. It is suggested that the local phenotype of peritubular fibroblasts in healthy and possibly also in injured kidneys may be controlled, at least in part, by the local tubular environment, conditioned by tubular metabolism and function.

Non-steroidal anti-inflammatory drugs decrease the quality of pleurodesis after mechanical pleural abrasion.

OBJECTIVE: Non-steroidal anti-inflammatory drugs (NSAIDs) are often applied for pain management after thoracic surgery. Since these drugs diminish collagen deposition through inhibition of the prostaglandin synthesis, we investigated their effects on adhesion formation after endoscopic mechanical pleural abrasion, which is often applied in the therapy of pneumothorax. METHODS: Mechanical pleural abrasion was performed unilaterally by the use of video-assisted thoracoscopic surgery technique in an established pig model. Ten animals (41.3+/-3.4 kg) were divided into a treatment group and a control group. In the treatment group, animals received 100 mg diclofenac (2 mg/kg body weight) orally daily for 3 weeks after surgery. At 3 weeks, all animals were sacrificed and efficacy of pleurodesis was macroscopically assessed by three independent reviewers blinded to the treatment of animals using a five-point severity pleurodesis score (from 0, no adhesions to 4, complete symphisis) and obliteration grade rating the distribution of adhesions (from 0, no adhesions to 4, adhesions in the whole chest). Microscopic evaluation was performed by two pathologists blinded to the study groups as well. A four-point score assessed the amount of collagen deposition (from 1, a few collagen fibers to 4, scar). RESULTS: Gross observation showed more dense adhesions in control animals with a median pleurodesis score of 3.67+/-1.0 in comparison to 2+/-2.2 in the treatment group (P = 0.01 *, Mann-Whitney non-parametric test). Distribution of adhesions was comparable in both groups with a median obliteration score of 3.67+/-1.3. Histopathologic examination showed a higher amount of collagen deposition in the control group, suggesting more dense adhesions, whereas in the treatment group there was loose granulation tissue (score of 4.0+/-0.8 vs. 2.3+/-1.0 in the treatment group, P = 0.06). The degree of inflammatory reaction was comparable in the two groups. CONCLUSIONS: Our results demonstrate that perioperative use of NSAIDs highly affects the quality of pleural adhesions obtained after mechanical abrasion in this pig model, which further suggests that these drugs should be avoided for pain management when a pleurodesis is performed.

Management of benign inverted sinonasal papilloma avoiding external approaches.

Even though endoscopic removal of inverted papillomas has gained popularity, many studies advocate supplementary external approaches. The impact of including the current surgical staging system into the pre-operative clinical and radiological assessment has not been systematically evaluated. We present our experience with total endoscopic management of inverted papillomas and compare the accuracy of the pre-operative predicted extent of surgery, with the actually performed surgery. From 1997 to 2005 data from 51 patients with inverted papillomas were prospectively collected and subsequently reviewed. All have been operated on endoscopically without an external approach. The overall recurrence rate was 3.9 per cent. Pre-operative prediction of extent of surgery was accurate in 26 of 51 (51 per cent). The main reasons for the inaccurate pre-operative prediction were the variable sizes and locations of the inverted papilloma bases, particularly in the maxillary sinus and the frontal recess. Our results encourage us to recommend endoscopic management as the standard treatment of benign inverted papillomas.

[Forensic and ballistic examination of the surface of gunshot wounds]. / A lösérülés felszínének orvos és fegyverszakértöi vizsgálatáról.

When investigating a gun-shot wound the forensic scientist gets the largest number of informations from the examination of the surface of it. After summarizing classical methods of the fire-arms examiner, authors report findings of the scanning electron microscopic examination of the entrance wound and the damaged clothing. Various alterations found characterize different factors of the shooting. Their presence and relation to each other may help to determine the fact of the shot itself and estimate the range at which the weapon was fired.

Transmission of scrapie by steel-surface-bound prions.

BACKGROUND: Prions are unusually resistant to conventional disinfection procedures. An electrode used intracerebrally on a Creutzfeldt-Jakob disease (CJD) patient transmitted the disease to two patients in succession and finally to a chimpanzee, despite attempted disinfection. Concerns that surgical instruments may transmit variant CJD have been raised by the finding of PrP(Sc), a surrogate marker for infectivity, in various tissues other than brain. MATERIALS AND METHODS: Stainless steel wire was exposed to scrapie-infected brain or brain homogenate, washed exhaustively and inserted into the brain of indicator mice to measure infectivity. RESULTS: A contact time of 5 min with scrapie-infected mouse brain suffices to render steel wire highly infectious and insertion of infectious wire into the brain of an indicator mouse for 30 min suffices to cause disease. Infectivity bound to wires persists far longer in the brain than when injected as homogenate, which can explain the extraordinary efficiency of wire-mediated infection. No detectable amounts of PrP could be eluted with NaOH, however the presence of PrP on infectious wires was demonstrated by chemiluminescence. Several recommended sterilisation procedures inactivated wire-bound mouse prions, but exposure to 10% formaldehyde was insufficient. CONCLUSIONS: Prions are readily and tightly bound to stainless steel surfaces and can transmit scrapie to recipient mice after short exposure times. This system mimics contaminated surgical instruments and will allow an assessment of sterilisation procedures.

Scrapie pathogenesis in subclinically infected B-cell-deficient mice.

Prion infections can present without clinical manifestations. B-cell deficiency may be a model for subclinical transmissible spongiform encephalopathy, since it protects mice from disease upon intraperitoneal administration of scrapie prions; however, a proportion of B-cell-deficient mice accumulate protease-resistant prion protein in their brains. Here, we have characterized this subclinical disease. In addition, we have studied the possibility that a neurotoxic factor secreted by B cells may contribute to pathogenesis.

Tonic stimulation of renin gene expression by nitric oxide is counteracted by tonic inhibition through angiotensin II.

This study was designed to examine the possible involvement of prostaglandins and nitric oxide (NO) in the renin stimulatory effect of angiotensin II (AngII) antagonists. To this end, plasma renin activities (PRAs) and renal renin mRNA levels were assayed in rats that were treated with the Ang-converting enzyme inhibitor ramipril or with the AngII AT1-receptor antagonist losartan. Ramipril and losartan increased PRA values from 7.5 +/- 1.6 to 86 +/- 6 and 78 +/- 22 ng of AngI per h per ml and renin mRNA levels from 112 +/- 9% to 391 +/- 20% and 317 +/- 10%, respectively. Inhibition of prostaglandin formation with indomethacin did not influence basal or ramipril-affected PRA. Basal renin mRNA levels also were unchanged by indomethacin, while increases in renin mRNA levels after ramipril treatment were slightly reduced by indomethacin. Inhibition of NO synthase by nitro-L-arginine methyl ester (L-NAME) reduced PRA values to 3.2 +/- 0.9, 34 +/- 13, and 12.1 +/- 2.7 ng of AngI per h per ml in control, ramipril-treated, and losartan-treated animals, respectively. Renin mRNA levels were reduced to 77 +/- 14% under basal conditions and ramipril- and losartan-induced increases in renin mRNA levels were completely blunted after addition of L-NAME. The AngII antagonists, furthermore, induced an upstream recruitment of renin-expressing cells in the renal afferent arterioles, which was also blunted by L-NAME. These findings suggest that renin mRNA levels are tonically increased by NO and that the action of NO is counteracted by AngII.

Effect of VEGF receptor inhibitor PTK787/ZK222584 [correction of ZK222548] combined with ionizing radiation on endothelial cells and tumour growth.

The vascular endothelial growth factor (VEGF) receptor is a major target for anti-angiogenesis-based cancer treatment. Here we report the treatment effect of ionizing radiation in combination with the novel orally bioavailable VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 on endothelial cell proliferation in vitro and with tumour xenografts in vivo. Combined treatment of human umbilical vein endothelial cells with increasing doses of PTK787/ZK222584 and ionizing radiation abrogated VEGF-dependent proliferation in a dose-dependent way, but inhibition of endothelial cell proliferation was not due to apoptosis induction. In vivo, a combined treatment regimen of PTK787/ZK222584 (4 x 100 mg/kg) during 4 consecutive days in combination with ionizing radiation (4 x 3 Gy) exerted a substantial tumour growth delay for radiation-resistant p53-dysfunctional tumour xenografts derived from SW480 colon adenocarcinoma cells while each treatment modality alone had only a minimal effect on tumour size and neovascularization. SW480 tumours from animals that received a combined treatment regimen, displayed not only an extended tumour growth delay but also a significant decrease in the number of microvessels in the tumour xenograft. These results support the model of a cooperative anti-tumoral effect of angiogenesis inhibitor and irradiation and show that the orally bioavailable VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 is suitable for combination therapy with irradiation.

Studies on prion replication in spleen.

Some of the early events following scrapie infection take place in the lymphoreticular system (LRS) and result in significant replication of prions in lymphoid organs. The identity of the cells in the LRS that produce prions and their role in neuroinvasion are still unknown. We find that in the spleen of scrapie-infected mice, prions are associated with T and B cells and to a somewhat lesser degree with the stroma, which contains the follicular dendritic cells (FDC's); curiously, no infectivity was found in lymphocytes from blood of the same mice. Thus, splenic lymphocytes either replicate prions or acquire them from another source. Studies on PrP knockout mice with ectopic expression of PrP restricted to only B or T lymphocytes suggest that neither of these by themselves are competent for prion replication. To determine whether B and T cells are able to pick up prions from other sources, irradiated wild-type mice were reconstituted with PrP-deficient lymphohaematopoietic stem cells. Following intraperitoneal inoculation of these mice, no infectivity was found on splenic lymphocytes whereas the stroma (comprising the radiation-resistant, PrP-expressing FDC's) contained prions. These results imply that splenic lymphocytes can acquire prions, possibly from FDC's, but only if they express PrP.

Tracking prions: the neurografting approach.

The physical nature of the agent that causes transmissible spongiform encephalopathies (the 'prion'), is the subject of passionate controversy. Investigation of it has benefited tremendously from the use of transgenic and knockout technologies. However, prion diseases present several other enigmas, including the mechanism of brain damage and how the affinity of the agent for the central nervous system is controlled. Here we show that such questions can be effectively addressed in transgenic and knockout systems, and that pathogenesis may be clarified even before we can be certain about the nature of the infectious agent. Availability of mice overexpressing the Prnp gene (which encodes the normal prion protein) and Prnp knockout mice allows for selective reconstitution experiments aimed at expressing PrP in specific portions of the brain or in selected populations of hemato- and lymphopoietic origin. We summarize how such studies can offer insights into how prions administered to peripheral sites can gain access to central nervous tissue, and into the molecular requirements for spongiform brain damage.

Prions and the lymphoreticular system.

Following intracerebral or peripheral inoculation of mice with scrapie prions, infectivity accumulates first in the spleen and only later in the brain. In the spleen of scrapie-infected mice, prions were found in association with T and B lymphocytes and to a somewhat lesser degree with the stroma, which contains the follicular dendritic cells (FDCs) but not with non-B, non-T cells; strikingly, no infectivity was found in lymphocytes from blood of the same mice. Transgenic PrP knockout mice expressing PrP restricted to either B or T lymphocytes show no prion replication in the lymphoreticular system. Therefore, splenic lymphocytes either acquire prions from another source or replicate them in dependency on other PrP-expressing cells. The essential role of FDCs in prion replication in spleen was shown by treating mice with soluble lymphotoxin-beta receptor, which led to disappearance of mature FDCs from the spleen and concomitantly abolished splenic prion accumulation and retarded neuroinvasion following intraperitoneal scrapie inoculation.