Pinus sylvestris L. and Syzygium aromaticum (L.) Merr. & L. M. Perry Essential Oils Inhibit Endotoxin-Induced Airway Hyperreactivity despite Aggravated Inflammatory Mechanisms in Mice.

Scots pine (SO) and clove (CO) essential oils (EOs) are commonly used by inhalation, and their main components are shown to reduce inflammatory mediator production. The aim of our research was to investigate the chemical composition of commercially available SO and CO by gas chromatography-mass spectrometry and study their effects on airway functions and inflammation in an acute pneumonitis mouse model. Inflammation was evoked by intratracheal endotoxin and EOs were inhaled three times during the 24 h experimental period. Respiratory function was analyzed by unrestrained whole-body plethysmography, lung inflammation by semiquantitative histopathological scoring, myeloperoxidase (MPO) activity and cytokine measurements. α-Pinene (39.4%) was the main component in SO, and eugenol (88.6%) in CO. Both SO and CO significantly reduced airway hyperresponsiveness, and prevented peak expiratory flow, tidal volume increases and perivascular edema formation. Meanwhile, inflammatory cell infiltration was not remarkably affected. In contrast, MPO activity and several inflammatory cytokines (IL-1ß, KC, MCP-1, MIP-2, TNF-α) were aggravated by both EOs. This is the first evidence that SO and CO inhalation improve airway function, but enhance certain inflammatory parameters. These results suggest that these EOs should be used with caution in cases of inflammation-associated respiratory diseases.

Complex Regulatory Role of the TRPA1 Receptor in Acute and Chronic Airway Inflammation Mouse Models.

The Transient Receptor Potential Ankyrin 1 (TRPA1) cation channel expressed on capsaicin-sensitive afferents, immune and endothelial cells is activated by inflammatory mediators and exogenous irritants, e.g., endotoxins, nicotine, crotonaldehyde and acrolein. We investigated its involvement in acute and chronic pulmonary inflammation using Trpa1 gene-deleted (Trpa1-/-) mice. Acute pneumonitis was evoked by intranasal Escherichia coli endotoxin (lipopolysaccharide: LPS) administration, chronic bronchitis by daily cigarette smoke exposure (CSE) for 4 months. Frequency, peak inspiratory/expiratory flows, minute ventilation determined by unrestrained whole-body plethysmography were significantly greater, while tidal volume, inspiratory/expiratory/relaxation times were smaller in Trpa1-/- mice. LPS-induced bronchial hyperreactivity, myeloperoxidase activity, frequency-decrease were significantly greater in Trpa1-/- mice. CSE significantly decreased tidal volume, minute ventilation, peak inspiratory/expiratory flows in wildtypes, but not in Trpa1-/- mice. CSE remarkably increased the mean linear intercept (histopathology), as an emphysema indicator after 2 months in wildtypes, but only after 4 months in Trpa1-/- mice. Semiquantitative histopathological scores were not different between strains in either models. TRPA1 has a complex role in basal airway function regulation and inflammatory mechanisms. It protects against LPS-induced acute pneumonitis and hyperresponsiveness, but is required for CSE-evoked emphysema and respiratory deterioration. Further research is needed to determine TRPA1 as a potential pharmacological target in the lung.

Effects of Thymus vulgaris L., Cinnamomum verum J.Presl and Cymbopogon nardus (L.) Rendle Essential Oils in the Endotoxin-induced Acute Airway Inflammation Mouse Model.

Thyme (TO), cinnamon (CO), and Ceylon type lemongrass (LO) essential oils (EOs) are commonly used for inhalation. However, their effects and mechanisms on inflammatory processes are not well-documented, and the number of in vivo data that would be important to determine their potential benefits or risks is low. Therefore, we analyzed the chemical composition and investigated the activity of TO, CO, and LO on airway functions and inflammatory parameters in an acute pneumonitis mouse model. The components of commercially available EOs were measured by gas chromatography-mass spectrometry. Airway inflammation was induced by intratracheal endotoxin administration in mice. EOs were inhaled during the experiments. Airway function and hyperresponsiveness were determined by unrestrained whole-body plethysmography on conscious animals. Myeloperoxidase (MPO) activity was measured by spectrophotometry from lung tissue homogenates, from which semiquantitative histopathological scores were assessed. The main components of TO, CO, and LO were thymol, cinnamaldehyde, and citronellal, respectively. We provide here the first evidence that TO and CO reduce inflammatory airway hyperresponsiveness and certain cellular inflammatory parameters, so they can potentially be considered as adjuvant treatments in respiratory inflammatory conditions. In contrast, Ceylon type LO inhalation might have an irritant effect (e.g., increased airway hyperresponsiveness and MPO activity) on the inflamed airways, and therefore should be avoided.

Morphological and Histological Effects of Radiofrequency and Laser (KTP and Nd:YAG) Treatment of the Inferior Turbinates in Animals.

The aim of this study was to evaluate the short and medium-term effects of radiofrequency (RF) and potassium titanyl phosphate (KTP) and neodymium-yttrium-aluminum garnet (Nd:YAG) laser treatment on the inferior turbinate mucosa in a porcine model. Following randomization, the inferior turbinates were treated either with RF submucosally or with the KTP or the Nd:YAG laser on the surface under videoendoscopic control. Tissue samples were taken at the end of postoperative weeks 1 and 6, and were evaluated macroscopically and histopathologically. Scanning electron microscopy was implemented to demonstrate the morphological changes in the respiratory epithelium. Six weeks following the RF procedure, the mucosa was intact in all cases, and the volume of the inferior turbinates was reduced in the majority of the cases. Although a volume reduction occurred in both laser groups, more complications associated with the healing procedure were noted. With hematoxylin and eosin and periodic acid-Schiff staining, intact epithelium, and submucosal glands remained after the RF procedures at the end of postoperative week 6. Following the KTP-laser intervention, necrotizing sialometaplasia and cartilage destruction occurred, and squamous metaplasia was also apparent in the Nd:YAG group. In both laser groups, dilated glands with excess mucus were seen. The scanning electron microscopic findings demonstrated that cilia were present in all cases. In conclusion, the medium-term macroscopic results were similar in all 3 groups, but the postoperative complications were less following the RF procedure. RF procedure is minimally invasive due to the submucosal intervention that leads to a painless, function preserving recovery.

Capsaicin-sensitive sensory nerves exert complex regulatory functions in the serum-transfer mouse model of autoimmune arthritis.

OBJECTIVE: The K/BxN serum-transfer arthritis is a widely-used translational mouse model of rheumatoid arthritis, in which the immunological components have thoroughly been investigated. In contrast, little is known about the role of sensory neural factors and the complexity of neuro-immune interactions. Therefore, we analyzed the involvement of capsaicin-sensitive peptidergic sensory nerves in autoantibody-induced arthritis with integrative methodology. METHODS: Arthritogenic K/BxN or control serum was injected to non-pretreated mice or resiniferatoxin (RTX)-pretreated animals where capsaicin-sensitive nerves were inactivated. Edema, touch sensitivity, noxious heat threshold, joint function, body weight and clinical arthritis severity scores were determined repeatedly throughout two weeks. Micro-CT and in vivo optical imaging to determine matrix-metalloproteinase (MMP) and neutrophil-derived myeloperoxidase (MPO) activities, semiquantitative histopathological scoring and radioimmunoassay to measure somatostatin in the joint homogenates were also performed. RESULTS: In RTX-pretreated mice, the autoantibody-induced joint swelling, arthritis severity score, MMP and MPO activities, as well as histopathological alterations were significantly greater compared to non-pretreated animals. Self-control quantification of the bone mass revealed decreased values in intact female mice, but significantly greater arthritis-induced pathological bone formation after RTX-pretreatment. In contrast, mechanical hyperalgesia from day 10 was smaller after inactivating capsaicin-sensitive afferents. Although thermal hyperalgesia did not develop, noxious heat threshold was significantly higher following RTX pretreatment. Somatostatin-like immunoreactivity elevated in the tibiotarsal joints in non-pretreated, which was significantly less in RTX-pretreated mice. CONCLUSIONS: Although capsaicin-sensitive sensory nerves mediate mechanical hyperalgesia in the later phase of autoantibody-induced chronic arthritis, they play important anti-inflammatory roles at least partially through somatostatin release.

Role of neurokinin 1 receptors in dextran sulfate-induced colitis: studies with gene-deleted mice and the selective receptor antagonist netupitant.

OBJECTIVE AND DESIGN: The function of the neurokinin 1 (NK1) receptor was investigated in the DSS-induced mouse colitis model using NK1 receptor-deficient mice and the selective antagonist netupitant. SUBJECTS: Colitis was induced by oral administration of 20 mg/ml DSS solution for 7 days in C57BL/6 and Tacr1 KO animals (n = 5-7). TREATMENT: During the induction, one-half of the C57BL/6 and Tacr1 KO group received one daily dose of 6 mg/kg netupitant, administered intraperitoneally, the other half of the group received saline, respectively. METHODS: Disease activity index (DAI), on the basis of stool consistency, blood and weight loss, was determined over 7 days. Histological evaluation, myeloperoxidase (MPO) measurement, cytokine concentrations and receptor expression analysis were performed on the colon samples. RESULTS: NK1 receptors are up-regulated in the colon in response to DSS treatment. DSS increased DAI, histopathological scores, BLC, sICAM-1, IFN-γ, IL-16 and JE in wildtype mice, which were significantly reduced in NK1 receptor-deficient ones. NK1 receptor antagonism with netupitant significantly diminished DAI, inflammatory histopathological alterations, BLC, IFN-γ, IL-13 and IL-16 in wildtype mice, but not in the NK1-deficient ones. MPO was similarly elevated and netupitant significantly decreased its activity in both groups. CONCLUSIONS: NK1 receptor antagonism could be beneficial for colitis via inhibiting different inflammatory mechanisms.

Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization are complementary techniques to detect cytogenetic abnormalities in multiple myeloma.

Multiple myeloma (MM) is a genetically heterogeneous disease with diverse clinical outcomes. Interphase fluorescence in situ hybridization (i-FISH) is the most commonly used approach to detect recurrent cytogenetic abnormalities in this malignancy. We aimed to assess the performance of multiplex ligation-dependent probe amplification (MLPA) to reveal copy number abnormalities (CNAs) in MM. Diagnostic bone marrow samples from 81 patients were analyzed using 42 MLPA probes for the following regions: 1p32-31, 1p21, 1q21.3, 1q23.3, 5q31.3, 12p13.31, 13q14, 16q12, 16q23, and 17p13. All samples were also screened by i-FISH for the presence of hyperdiploidy, deletion/monosomy of chromosome 13, deletion of TP53, disruption of the immunoglobulin heavy-chain gene, t(4;14), t(11;14), t(14;16), t(8;14), gain of 5q and abnormalities of chromosome 1. A total of 245 alterations were detected in 79 cases (98%). Investigating the same aberrations, the two methods showed a congruency of higher than 90%. A low proportion of cells with the relevant abnormality, focal CNAs and unmatched probes were responsible for the discrepancies. MLPA revealed 95 CNAs not detected by i-FISH providing additional information in 53 cases (65%). Scrutiny of CNAs on chromosome 1, using more than 20 probes, revealed significant heterogeneity in size and location, and variable intra-chromosomal and intra-clonal rates of loss or gain. Our results suggest that MLPA is a reliable high-throughput technique to detect CNAs in MM. Since balanced aberrations are key to prognostic classification of this disease, MLPA and i-FISH should be applied as complementary techniques in diagnostic pathology.

ASSOCIATION OF TEMPORAL LOBE INFLAMMATORY LEUKOENCEPHALOPATHY WITH TWO B CELL MALIGNANCIES.

Identification of etiological connections among virtually distinct diseases in a patient may be sometimes challenging. We report a unique case with two B cell malignancies and an inflammatory leukoencephalopathy. Three days prior to admission, the elderly male patient developed fatigue, headaches, recurrent vomiting, memory disturbances, depression and somnolence. Clinical, laboratory and imaging evaluations as well as post mortem histological studies were performed. Simultaneous presence of primary central nervous system B cell lymphoma, temporal lobe inflammatory leukoencephalopathy and multiple (smoldering) myeloma, was revealed by the detailed work up in the treatment-naïve patient. Based on recent data from genomic studies, we propose that a sequential evolution of molecular pathology lead to the co-occurrence of multiple myeloma and primary central nervous system B cell lymphoma in this patient, and interpret the development of the temporal lobe leukoencephalopathy as a likely paraneoplastic complication of smoldering myeloma.

Case report: Initial atypical skeletal symptoms and dental anomalies as first signs of Gardner syndrome: the importance of genetic analysis in the early diagnosis.

Background: Gardner syndrome is a rare genetic cancer predisposition disorder characterized by intestinal polyposis, multiple osteomas, and soft and hard tissue tumors. Dental anomalies are present in approximately 30%-70% of patients with Gardner syndrome and can be discovered during routine dental examinations. However, sometimes the diagnosis is challenging due to the high clinical variability and incomplete clinical picture. Herein, we report a family with various dental and bone anomalies, in which the definitive diagnosis was established with the help of a comprehensive genetic analysis based on state-of-the-art next-generation sequencing technology. Case presentation: A 17-year-old female index patient presented with dental (caries, impacted, retained and anteriorly located teeth) and atypical bone anomalies not resembling Gardner syndrome. She was first referred to our Genetic Counselling Unit at the age of 11 due to an atypical bone abnormality identified by a panoramic X-ray. Tooth 3.6 was surgically removed and the histopathology report revealed a Paget's disease-like bone metabolic disorder with mixed osteoblastic and osteoclastic activity of the mandible. A small lumbar subcutaneous tumor was discovered by physical examination. Ultrasound examination of the tumor raised the possibility of a soft tissue propagation of chondromatosis. Her sister, 2 years younger at the age of 14, had some benign tumors (multiple exostoses, odontomas, epidermoid cysts) and impacted teeth. Their mother had also skeletal symptoms. Her lower teeth did not develop, the 9th-10th ribs were fused, and she complained of intermittent jaw pain. A cranial CT scan showed fibrous dysplasia on the cranial bones. Whole exome sequencing identified a heterozygous pathogenic nonsense mutation (c.4700C>G; p.Ser1567*) in the APC gene in the index patient's DNA. Targeted sequencing revealed the same variant in the DNA of the other affected family members (the sister and the mother). Conclusion: Early diagnosis of this rare, genetically determined syndrome is very important, because of the potentially high malignant transformation of intestinal polyps. Dentists should be familiar with the typical maxillofacial features of this disorder, to be able to refer patients to genetic counseling. Dental anomalies often precede the intestinal polyposis and facilitate the early diagnosis, thereby increasing the patients' chances of survival. Genetic analysis may be necessary in patients with atypical phenotypic signs.

Clinicopathological analysis of diffuse large B-cell lymphoma using molecular biomarkers: a retrospective analysis from 7 Hungarian centers.

Background: The clinical and genetic heterogeneity of diffuse large B-cell lymphoma (DLBCL) presents distinct challenges in predicting response to therapy and overall prognosis. The main objective of this study was to assess the application of the immunohistochemistry- and interphase fluorescence in situ hybridization (FISH)-based molecular markers in the diagnosis of DLBCL and its prognostic value in patients treated with rituximab-based immunochemotherapy. Methods: This is a multicenter, retrospective study, which analyzed data from 7 Hungarian hematology centers. Eligible patients were adults, had a histologically confirmed diagnosis of DLBCL, were treated with rituximab-based immunochemotherapy in the first line, and had available clinicopathological data including International Prognostic Index (IPI). On the specimens, immunohistochemistry and FISH methods were performed. Germinal center B-cell like (GCB) and non-GCB subtypes were classified by the Hans algorithm. Outcomes included overall survival (OS), event-free survival (EFS), and EFS at 2 years (EFS24). For survival analysis, we used Kaplan-Meier curves with the log-rank test and multivariate Cox regression. Results: A total of 247 DLBCL cases were included. Cases were positive for MYC, BCL2, BCL6, and MUM1 expression in 52.1%, 66.2%, 72.6%, and 77.8%, respectively. BCL6 translocation, BCL2 gene copy number (GCN) gain, IGH::MYC translocation, MYC GCN gain, IGH::BCL2 translocation, and BCL6 GCN gain were detected in 21.4%, 14.1%, 7.3%, 1.8%, 7.3%, and 0.9%, respectively. At a median follow-up of 52 months, 140 patients (56.7%) had disease progression or relapse. The Kaplan-Meier estimate for EFS24 was 56.2% (CI: 50.4-62.8%). In univariate analysis, only IPI and BCL6 expression were significant predictors of both OS and EFS, whereas MUM1 predicted EFS only. In multivariate analysis, the IPI score was a significant independent negative, whereas MIB-1 and BCL6 protein expressions were significant independent positive predictors of both OS and EFS. Conclusion: In our study, we found that only IPI, BCL6 protein expression and MIB-1 protein expression are independent predictors of survival outcomes in DLBCL. We did not find any difference in survival by GCB vs. non-GCB subtypes. These findings may improve prognostication in DLBCL and can contribute to designing further research in the area.

Composite urinary reservoir in dogs: histological findings.

PURPOSE: This animal study was designed to investigate whether the composite urinary reservoir might lessen the premalignant histological alterations observed after bladder augmentation performed with a gastric segment or large bowel. MATERIALS AND METHODS: Composite urinary reservoirs were created using gastric and colonic segments simultaneously in 8, 3-month-old female beagle dogs by augmenting half the native bladder. Two dogs with gastrocystoplasty and 2 with colocystoplasty served as controls. Biopsies were taken from the native bladder, and the gastric and colonic segments at augmentation, and endoscopically 4 and 8 months postoperatively. The dogs were sacrificed and open biopsied 12 months postoperatively. Tissue specimens were examined with routine hematoxylin and eosin, reaction and immunohistological staining for PCNA. RESULTS: At the creation of composite reservoir and gastrocoloplasty or colocystoplasty all specimens showed normal histology. At 12 months postoperatively dysplasia was found in 1 gastric segment, 2 native bladders and 3 colonic segments in the composite reservoir group. There was a single carcinoma in situ in 1 gastric segment in the composite reservoir group. In the control groups 1 colonic segment and 1 native bladder dysplasia were detected at the end of 12-month followup. There was an in situ carcinoma in 1 gastric segment in the composite reservoir. CONCLUSIONS: A composite reservoir did not decrease premalignant changes in dogs during 12 months of followup. Laboratory investigations, molecular studies and longer followup are needed to approach the question of early malignant alterations after augmentation cystoplasty in animals and patients.

Impaired defense mechanism against inflammation, hyperalgesia, and airway hyperreactivity in somatostatin 4 receptor gene-deleted mice.

We have shown that somatostatin released from activated capsaicin-sensitive nociceptive nerve endings during inflammatory processes elicits systemic anti-inflammatory and analgesic effects. With the help of somatostatin receptor subtype 4 gene-deleted mice (sst(4)(-/-)), we provide here several lines of evidence that this receptor has a protective role in a variety of inflammatory disease models; several symptoms are more severe in the sst(4) knockout animals than in their wild-type counterparts. Acute carrageenan-induced paw edema and mechanical hyperalgesia, inflammatory pain in the early phase of adjuvant-evoked chronic arthritis, and oxazolone-induced delayed-type hypersensitivity reaction in the skin are much greater in mice lacking the sst(4) receptor. Airway inflammation and consequent bronchial hyperreactivity elicited by intranasal lipopolysaccharide administration are also markedly enhanced in sst(4) knockouts, including increased perivascular/peribronchial edema, neutrophil/macrophage infiltration, mucus-producing goblet cell hyperplasia, myeloperoxidase activity, and IL-1beta, TNF-alpha, and IFN-gamma expression in the inflamed lung. It is concluded that during these inflammatory conditions the released somatostatin has pronounced counterregulatory effects through sst(4) receptor activation. Thus, this receptor is a promising novel target for developing anti-inflammatory, analgesic, and anti-asthmatic drugs.

Should the ossicle be denuded prior to the application of glass ionomer cement? An experimental study on rabbit.

The objective of this study was to assess whether denudation of the auditory ossicle prior to the application of glass ionomer cement (GIC) durably strengthens the adhesion between bone and GIC. The tympanic bullas of 34 rabbits were opened bilaterally. The mucosa was removed from the lateral surface of the right-side incudi with a diamond burr, while the left-side incudi were left intact. GIC was then applied bilaterally to the lateral surface of the incudi of 30 of these rabbits which were subsequently killed 1, 3, 7, 14, 21, 30, 60, 90, 180 or 365 days postoperatively. The 4 sham-operated animals were killed on day 1, 7, 30 or 365. The incudi were removed and processed for histological evaluation. On exploration, the cement was visible on the incus within the tympanic bulla in all 30 GIC-treated animals. During surgical removal, the GIC was separated from the incus in 3 ears. Histological examination further revealed separation in 5 ears after processing. All 8 separations occurred in the right (not denuded) ears, and at least 60 days postoperatively. The difference between the two sides in the number of separations was significant (p < 0.05). The initial inflammatory reaction elicited by the surgical trauma to the right-side ossicles had substantially decreased by day 7. No foreign body reaction was observed and the GIC became overgrown with mucosa by day 60. In conclusion, the GIC proved biocompatible, and preliminary denudation of the ossicle resulted in stronger and more durable bone-GIC adhesion.

Sex chromosome changes after sex-mismatched allogeneic bone marrow transplantation can mislead the chimerism analysis.

A 12-year-old male with pre-B-cell acute lymphoblastic leukemia with cryptic BCR/ABL rearrangement underwent sex-mismatched allogeneic bone marrow transplantation (allo-BMT). Contradictory results were provided by various chimerism analyses 3 months later. Y-chromosome-specific quantitative polymerase chain reaction and sex chromosome-specific interphase fluorescence in situ hybridization (i-FISH) showed complete donor chimerism. Analysis of autosomal short tandem repeats (A-STR), BCR/ABL i-FISH test, and X-STR haplotype indicated relapse. Metaphase-FISH and combined BCR/ABL and sex chromosome-specific i-FISH patterns revealed loss of the Y-chromosome and duplication of the X-chromosome in the host cells. Sex chromosome changes after allo-BMT can cause significant difficulties in chimerism analysis.

Lipid and protein tumor markers for head and neck squamous cell carcinoma identified by imaging mass spectrometry.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. To improve pre- and post-operative diagnosis and prognosis novel molecular markers are desirable. Here we used MALDI imaging mass spectrometry (IMS) and immunohistochemistry (IHC) to seek tumor specific expression of proteins and lipids in HNSCC samples. Among low molecular weight proteins visualized, S100A8 and S100A9 were found to be expressed in the regions of tumor tissue but not in the surrounding healthy stroma of a post-operative microdissected tissue. Marker potential of S100A8 and S100A9 was confirmed by immunohistochemistry of paraffin-embedded pathological samples. Imaging lipids showed a remarkable depletion of lysophosphatidylcholine species LPC[16:0], LPC[18:2] and, in parallel, accumulation of major glycerophospholipid species PE-P[36:4], PC[32:1], PC[34:1] in neoplastic areas. This was confirmed by shotgun lipidomics of dissected healthy and tumor tissue sections. A combination of the negative (LPC[16:0]) and positive (PC[32:1], PC[34:1]) markers was also applicable to uncover tumorous character of a pre-operative biopsy. Furthermore, marker potential of lysophospholipids was supported by elevated expression levels of the lysophospholipid degrading enzyme lysophospholipase A1 (LYPLA1) in the tumor regions of paraffin-embedded HNSCC samples. Finally, experimental evidence of 3D cell spheroid tests showed that LPC[16:0] facilitates HNSCC invasion, implying that HNSCC progression in vivo may be dependent on lysophospholipid supply. Altogether, a series of novel proteins and lipid species were identified by IMS and IHC screening, which may serve as potential molecular markers for tumor diagnosis, prognosis, and may pave the way to better understand HNSCC pathophyisiology.

[Molecular cytogenetic analyses of patients with plasma cell myeloma in Tolna and Baranya counties in Hungary]. / Molekuláris citogenetikai vizsgálatok Baranya és Tolna megye plazmasejtes myelomában szenvedo betegein.

Introduction: Plasma cell myeloma is a hematological malignancy with heterogeneous genomic landscape and diverse clinical course. Recurrent chromosomal and subchromosomal aberrations commonly occur in this entity and are associated with the pathogenesis and progression of the disease. The identification of these alterations aids genetic characterization, classification and prognostication of patients. Aim: Molecular cytogenetic investigations of plasma cell myeloma patients treated at the University of Pécs Clinical Center and János Balassa County Hospital of Tolna County, Szekszárd, between 2005 and 2018 were evaluated in our study. Method: 231 patients were screened for genetic aberrations using fluorescence in situ hybridization. Translocations involving the immunoglobulin heavy chain gene, losses of 1p and 17p chromosome arms, gains of 1q chromosome arm and unbalanced aberrations of chromosome 13 were investigated. Losses and gains of 1p, 1q, 5q, 12p, 13q, 16q and 17p chromosome arms were analyzed using multiplex ligation-dependent probe amplification in 42 patients. During the investigated period, 116 bone marrow karyotyping was also performed. Results: In total, 233 genetic aberrations were identified using our targeted approaches; the frequency of specific aberrations correlated with data of the recent literature. Concordance of results gained by fluorescence in situ hybridization and multiplex ligation-dependent probe amplification was 96.2% by analyzing the same chromosome arms. The latter technique revealed 21 additional genetic aberrations in 16/42 patient samples (38%) as compared to fluorescence in situ hybridization. Conclusions: Our results suggest that the combined application of the two molecular cytogenetic methods may facilitate a more detailed characterization of genetic aberrations of plasma cell myeloma patients in Hungary. Orv Hetil. 2019; 160(24): 944-951.

[Impact of minimal residual disease detection after treatment of multiple myeloma]. / A minimális residualis betegség vizsgálatának jelentosége myeloma multiplex kezelése után.

INTRODUCTION: Prognostic impact of the detection of minimal residual disease (MRD) in multiple myeloma (MM) has been confirmed in numerous studies. AIM: Retrospective examination of our patient database (107 newly diagnosed multiple myelomas between 2007 and 2017). Flow cytometry (FCM) was performed as MRD assessment. METHOD: MRD assessment was performed in 56 patients (median age: 68 years), after induction treatment of multiple myeloma. The treatment contained bortezomib in 91%, autologous haematopoetic stem cell transplantation (ASCT) was perfomed in 50%. MRD detection was performed on bone marrow samples, predominantly in our hospital (BD FACScan, 3 colour, panel: CD38, CD138, CD19, CD45, CD56, CD28, CD117, cyKappa, cyLambda, 100 000 events). STATISTICAL ANALYSIS: SPSS 13.0. RESULTS: The progression-free survival (PFS) and the overall survival (OS) were significantly longer in MRD negative (n = 22) patients (PFS: 54 months, OS: 79% after 5 years) than MRD positive patients (n = 34, PFS: 22 months, OS 21% after 5 years, p = 0.001). Patients achieving complete response (CR) (n = 29) have different PFS (MRD negative CR: 60 months, MRD positive CR: 21 months, p<0.001). Patents achiving MRD negative very good partial response (n = 5) have similar PFS (54 months) as patients with MRD negative CR. The longest PFS (68 months) was observed in MRD negative patients, after ASCT (n = 11), while the PFS was significantly (p<0.001) shorter in patients who were MRD positive after ASCT (n = 18, PFS: 25 months), similarly in MRD positive patients without ASCT (n = 15, PFS 21 months). Cox regression analysis (stage, cytogenetic risk, ASCT) confirmed that MRD is an independent prognostic factor of PFS and OS. We did not find significant relationship between MRD and stage, cytogenetic risk, number of treatment cycles, ASCT. CONCLUSIONS: The depth of response after induction treatment of MM is an independent predictor of survival. MRD assessment with FCM is recommended to define response. Consideration of maintenance treatment in MRD positive patients and eradication of MRD are also recommended. Orv Hetil. 2019; 160(13): 502-508.

Re-evaluation of histological findings after colocystoplasty and gastrocystoplasty.

INTRODUCTION: The potential of malignant transformation and its risk factors after bladder augmentation performed in childhood are still unknown. The necessity of surveillance cystoscopies and biopsies has been questioned in the past decade. OBJECTIVE: In a previous study, the authors did not detect any malignancy after colocystoplasty (CCP) or gastrocystoplasty (GCP) during the short-term follow-up, however, various alterations of the mucosa were found. A correlation between the nature of histological changes and the frequency of bacterial colonization after CCP were also found. The authors hypothesized that a longer-term follow-up of their patients would reveal an increase in pathological change or show stronger association between the histological alteration, bacterial colonization, and/or stone formation. PATIENTS AND METHODS: Thirty-five patients (20 cases of colocystoplasties - CCPs; 15 cases of gastrocystoplasties - GCPs) participated in the study published in 2002. All patients were followed biannually with endoscopic assessment and biopsies. Two independent pathologists, evaluated regular biopsies from the native bladder, from the segment used for augmentation and from the anastomosis line. Etiology, frequency of positive urine cultures, and stone events were recorded and compared with histological findings between groups and with the previously published results. RESULTS: Continuous surveillance allowed the follow-up of 30 patients (CCP 19/20, GCP 11/15) for 20 and 15 years. No malignancies were identified. Results of biopsies showed significant difference between groups (summarized in the tables). Chronic inflammatory changes were found following both types of augmentations, but they were more common in the urothelium following GCP and more common in the colonic mucosa following CCP. The rate of metaplastic lesions was higher after gastrocystoplasty (GCP). Significant association was found between the etiologic factor and the nature of histological change after CCP, as metaplastic lesions occurred only in patients with bladder exstrophy. Stones occurred more frequently in exstrophy patients as well. The nature of the histological changes did not correlate with positive urine cultures in either of the groups. Significantly higher incidence of bacterial colonization and stone occurrence were found after CCP. CONCLUSIONS: Long-term follow-up of the patients failed to reveal an increase in pathological changes, and no malignancies were observed. According to the results of this study, etiology of bladder dysfunction and the type of augmentation might influence the histological alterations after augmentation cystoplasty. The efficiency of surveillance cystoscopies and biopsies are low. The present data suggest that surveillance cystoscopy and biopsy should not be routinely performed, and should be done only if the symptoms are suspicious for malignancy.

Automated FISH analysis using dual-fusion and break-apart probes on paraffin-embedded tissue sections.

Detecting balanced translocations using tissue sections plays an important diagnostic role in cases of hematological malignancies. Manual scoring is often problematic due to truncation and overlapping of nuclei. Reports have described automated analysis using primarily tile sampling. The aim of this study was to investigate an automated fluorescent in situ hybridization analysis method using grid sampling on tissue sections, and compare the performance of dual-fusion (DF) and break-apart (BA) probes in this setting. Ten follicular, 10 mantle cell lymphoma, and 10 translocation-negative samples were used to set the threshold of false positivity using IGH/CCND1, IGH/BCL-2 DF, and IGH BA probes. The cut-off distances of red and green signals to define fusion signals were 0.5, 1.0, and 1.2 mum for the IGH/CCND1, IGH/BCL-2 DF, and IGH BA probes, respectively. The mean false positivity of grid units was 5.3, 11.4, and 28.1%, respectively. Ten to 14 additional samples analyzed blindly and were correctly classified using each probe. Discriminating positive and negative samples using automated analysis and grid sampling was possible with each probe, although different definitions of fusion signals were required due to the different physical distances between the DNA probes. Using the DF probes resulted in lower false positivity, which was less affected by signal numbers per grid units.

Inhibitory effects of synthetic somatostatin receptor subtype 4 agonists on acute and chronic airway inflammation and hyperreactivity in the mouse.

Somatostatin released from activated capsaicin-sensitive afferents of the lung inhibits inflammation and related bronchial hyperreactivity presumably via somatostatin 4 receptors (sst(4)). The aim of this study was to examine the effects of TT-232, a heptapeptide sst(4)/sst(1) receptor agonist and J-2156, a high affinity sst(4) receptor-selective peptidomimetic agonist in airway inflammation models. Acute pneumonitis was evoked by intranasal lipopolysaccharide 24 h before measurement. Chronic inflammation was induced by ovalbumin inhalation on days 28, 29 and 30 after i.p. sensitization on days 1 and 14. Semiquantitative histopathological scoring was based on perivascular/peribronchial oedema, neutrophil/macrophage infiltration, goblet cell hyperplasia in the acute model and eosinophil infiltration, mucosal oedema, mucus production and epithelial cell damage in chronic inflammation. Myeloperoxidase activity of the lung was measured spectrophotometrically to quantify granulocyte accumulation and the broncoalveolar lavage fluid was analysed by flow cytometry. Carbachol-induced bronchoconstriction was assessed by unrestrained whole body plethysmography and its calculated indicator, enhanced pause (Penh) was determined. TT-232 and J-2156 induced similar inhibition on granulocyte recruitment and histopathological changes in both models, although macrophage infiltration in LPS-induced inflammation was unaltered by either compounds. Both agonists diminished inflammatory airway hyperresponsiveness. Since their single administration after the development of the inflammatory reactions also inhibited carbachol-induced bronchoconstriction, somatostatin sst(4) receptor activation on bronchial smooth muscle cells is likely to be involved in their anti-hyperreactivity effect. These results suggest that stable, somatostatin sst(4) receptor-selective agonists could be potential candidates for the development of a completely novel group of anti-inflammatory drugs for the treatment of airway inflammation and hyperresponsiveness.