Identification of pathogens causing invasive fungal rhinosinusitis in surgical biopsies using polymerase chain reaction.

BACKGROUND: Invasive fungal rhinosinusitis is associated with high morbidity and mortality. Rapid pathogen identification is mandatory, but fresh tissue is not always available. A polymerase chain reaction method was designed in order to detect fungi in formalin-fixed paraffin-embedded samples. This was applied to a retrospective series of tissue biopsies from Thai patients with invasive fungal rhinosinusitis. METHODS: Tissue blocks from 64 cases yielded adequate DNA. Three sequential polymerase chain reaction were performed: ZP3 (housekeeping gene) and panfungal polymerase chain reactions, and a differentiating polymerase chain reaction based on the 5.8s ribosomal RNA and internal transcribed spacer 2 regions. The polymerase chain reaction products were then sequenced. RESULTS: Polymerase chain reaction identified a fungal pathogen in 20 of 64 cases (31 per cent). Aspergillus species was the most common cause of invasive fungal rhinosinusitis (nine cases). Other causes included candida (n = 4), cladosporium (n = 4), mucor (n = 1), alternaria (n = 1) and dendryphiella (n = 1) species. CONCLUSION: Polymerase chain reaction can provide rapid identification of fungal pathogens in paraffin-embedded tissue, enabling prompt treatment of invasive fungal rhinosinusitis.

Detection of global hypermethylation in well-differentiated thyroid neoplasms by immunohistochemical (5-methylcytidine) analysis.

PURPOSE: While global hypomethylation of DNA has been found in several malignancies, studies on thyroid tumours have shown controversial results using different techniques. To help resolve this issue, we assessed methylation status using two different techniques in papillary thyroid carcinomas (PTC) and follicular adenomas (FA) and carcinomas (FTC), comparing adjacent non-neoplastic thyroid tissue. METHODS: A series of 15 FA, 18 FTC and 17 PTC were assessed by: (1) measurement of methylation levels of long interspersed nuclear elements (LINE-1) using a combined bisulfite restriction analysis polymerase chain reaction protocol and (2) immunostaining with an anti-5-methylcytidine antibody that detects methylated DNA regardless of the DNA sequence. Immunostaining was scored by image analysis. RESULTS: Methylation levels of LINE-1 in FA, FTC and PTC were not significantly different from adjacent normal tissue. There was no significant difference in methylation levels of LINE-1 between FA, FTC and PTC (p = 0.44). By immunohistochemical staining for methylation, the 5-methylcytidine score was significantly higher in tumours than in normal tissue counterparts, for FA (p < 0.001), FTC (p = 0.04) and PTC (p = 0.02). PTC showed the highest 5-methylcytidine expression amongst all tumours which was significantly different from FTC (p = 0.015), but not FA (p = 0.09). There was no correlation in methylation level between LINE-1 and 5-methylcytidine scores for each group and overall. CONCLUSIONS: Well-differentiated thyroid neoplasms (FA, FTC and PTC) were not found by two independent methods to undergo global hypomethylation as part of an oncogenic sequence from normal tissue to carcinoma. Instead, hypermethylation was detected in all types of tumours, implying that this epigenetic event may contribute to oncogenic development of thyroid neoplasms (both benign and malignant).

Antibody mediated rejection associated with complement factor h-related protein 3/1 deficiency successfully treated with eculizumab.

Antibody mediated rejection (AMR) activates the classical complement pathway and can be detrimental to graft survival. AMR can be accompanied by thrombotic microangiopathy (TMA). Eculizumab, a monoclonal C5 antibody prevents induction of the terminal complement cascade (TCC) and has recently emerged as a therapeutic option for AMR. We present a highly sensitized 13-year-old female with end-stage kidney disease secondary to spina bifida-associated reflux nephropathy, who developed severe steroid-, ATG- and plasmapheresis-resistant AMR with TMA 1 week post second kidney transplant despite previous desensitization therapy with immunoglobulin infusions. Eculizumab rescue therapy resulted in a dramatic improvement in biochemical (C3; creatinine) and hematological (platelets) parameters within 6 days. The patient was proven to be deficient in complement Factor H-related protein 3/1 (CFHR3/1), a plasma protein that regulates the complement cascade at the level of C5 conversion and has been involved in the pathogenesis of atypical hemolytic uremic syndrome caused by CFH autoantibodies (DEAP-HUS). CFHR1 deficiency may have worsened the severe clinical progression of AMR and possibly contributed to the development of donor-specific antibodies. Thus, screening for CFHR3/1 deficiency should be considered in patients with severe AMR associated with TMA.

Analysis of segmental duplications, mouse genome synteny and recurrent cancer-associated amplicons in human chromosome 6p21-p12.

It has been proposed that regions of microhomology in the human genome could facilitate genomic rearrangements, copy number transitions, and rapid genomic change during tumor progression. To investigate this idea, this study examines the role of repetitive sequence elements, and corresponding syntenic mouse genomic features, in targeting cancer-associated genomic instability of specific regions of the human genome. Automated database-mining algorithms designed to search for frequent copy number transitions and genomic breakpoints were applied to 2 publicly-available online databases and revealed that 6p21-p12 is one of the regions of the human genome most frequently involved in tumor-specific alterations. In these analyses, 6p21-p12 exhibited the highest frequency of genomic amplification in osteosarcomas. Analysis of repetitive elements in regions of homology between human chromosome 6p and the syntenic regions of the mouse genome revealed a strong association between the location of segmental duplications greater than 5 kilobase-pairs and the position of discontinuities at the end of the syntenic region. The presence of clusters of segmental duplications flanking these syntenic regions also correlated with a high frequency of amplification and genomic alteration. Collectively, the experimental findings, in silico analyses, and comparative genomic studies presented here suggest that segmental duplications may facilitate cancer-associated copy number transitions and rearrangements at chromosome 6p21-p12. This process may involve homology-dependent DNA recombination and/or repair, which may also contribute towards the overall plasticity of the human genome.

PCR detection of Mycobacterium tuberculosis in necrotising non-granulomatous lymphadenitis using formalin-fixed paraffin-embedded tissue: a study in Thai patients.

BACKGROUND: Necrotising non-granulomatous lymphadenitis can be observed in several conditions, most notably infection (including tuberculosis, yersiniosis and nocardiasis), Kikuchi-Fujimoto disease and systemic lupus erythematosus. AIMS: To evaluate the role of PCR in the detection of Mycobacterium tuberculosis in necrotising non-granulomatous lymphadenitis in Thai patients using formalin-fixed paraffin-embedded tissue. METHODS: 35 patient samples showing necrotising non-granulomatous lymphadenitis were subjected to PCR for detection of the IS6110 sequence of M tuberculosis. For comparison, sections were visually assessed for acid-fast bacilli using the Ziehl-Neelsen stain. RESULTS: Among 35 cases of necrotising non-granulomatous lymphadenitis, a conclusive diagnosis could be reached in 23 cases: 15 cases of Kikuchi-Fujimoto disease, 6 of tuberculosis and 2 of systemic lupus erythematosus. Of the 6 cases of tuberculous lymphadenitis, 4 (66.6%) were detected by PCR in formalin-fixed paraffin-embedded tissue samples. PCR was positive in 6/12 of the remaining cases (50%) in which a definitive diagnosis could not be reached by other methods. CONCLUSION: Using PCR, a significant percentage (28%) of cases of necrotising non-granulomatous lymphadenitis in this study could be attributed to M tuberculosis. PCR for identification of the organism can be extremely helpful in confirming a diagnosis of tuberculosis when Ziehl-Neelsen staining is negative.

Cisplatin treatment increases survival and expansion of a highly tumorigenic side-population fraction by upregulating VEGF/Flt1 autocrine signaling.

The cellular and molecular mechanisms of tumor progression following chemotherapy are largely unknown. Here, we demonstrate that cisplatin (CDDP) treatment upregulates VEGF and Flt1 expression leading to the survival and expansion of a highly tumorigenic fraction of side-population (SP) cells in osteosarcoma (HOS), neuroblastoma (SK-N-BE2) and rhabdomyosarcoma (RH-4) cell lines. In all three lines, we show that CDDP treatment increases levels of VEGF and Flt1 expression, and induces enhanced clonogenic capacity and increased expression of the 'stemness'-associated genes Nanog, Bmi-1 and Oct-4 in the SP fraction. In HOS, these changes are associated with the transformation of a non-tumorigenic osteosarcoma SP fraction to a highly tumorigenic phenotype. Inhibition of Flt1 led to complete reduction of tumorigenicity in the HOS SP fraction, and reduction of clonogenic capacity and expression of stemness genes in the SK-N-BE(2) and RH-4 SP fractions. Treatment with U0126, a specific inhibitor of MAPK/ERK1,2 completely downregulates CDDP-induced VEGF and Flt1 expression and induction/expansion of SP fraction in all three cell lines, indicating that these effects are mediated through MAPK/ERK1,2 signaling. In conclusion, we report a novel mechanism of CDDP-induced tumor progression, whereby the activation of VEGF/Flt1 autocrine signaling leads to the survival and expansion of a highly tumorigenic SP fraction.

Increased mast cell density in haemorrhoid venous blood vessels suggests a role in pathogenesis.

INTRODUCTION: Haemorrhoids are an abnormal, tortuous dilatation of the arteriovenous plexus of the anus. Although increased resting anorectal pressure is deemed to be a major initiating factor, a thorough understanding of the pathogenesis is still lacking. Mast cells, through release of granules, can affect local vessels with respect to changes in calibre, changes in permeability and thrombosis. Thus, mast cells could play a role in haemorrhoid pathophysiology, although this has not been previously investigated. METHODS: 48 cases of haemorrhoids were retrospectively collected at King Chulalongkorn Memorial Hospital, with normal anorectal tissue from surgically-removed colorectal cancer serving as controls. Mast cells were identified by toluidine blue staining and quantitated around venous vessels. RESULTS: Mast cells around haemorrhoidal vessels were significantly more numerous than in normal specimens (p-value is less than 0.001). Similar values were found for haemorrhoids showing chronic changes and those in a more acute stage. CONCLUSION: These findings support the hypothesis that mast cells may play a role in the pathophysiology of haemorrhoids. Mast cells appear to participate equally in the early and later stages of these lesions. Mast cells are known to affect local vascular conditions through release of their chemical mediators and cytokines, and may influence haemorrhoid symptomatology and progression at this level.

Clinical features and outcome of pediatric Wegener's granulomatosis.

OBJECTIVE: Wegener's granulomatosis (WG) is a predominantly small-vessel vasculitis associated with antineutrophil cytoplasmic antibodies (ANCAs). There are few reports describing its clinical features and outcome in children. We report on the experience at a single tertiary referral center over 21 years. METHODS: We conducted a retrospective chart review of all patients diagnosed with WG at The Hospital for Sick Children between 1984 and 2005. RESULTS: Twenty-five patients were identified. Median age at diagnosis and median followup were 14.5 years and 32.7 months, respectively. Male-to-female ratio was 1:4. Median duration of symptoms before diagnosis was 2 months. Of 22 patients, 21 were ANCA positive during their disease course (classic ANCA 78.9%). Constitutional symptoms were the most common clinical feature at presentation (24 of 25). Glomerulonephritis was present in 22 patients at presentation. Only 1 of 11 patients who presented with or developed renal impairment had normalization of serum creatinine. Upper airway involvement occurred in 21 patients at presentation and 24 over followup; only 1 had subglottic stenosis. Twenty patients had initial pulmonary involvement, most commonly nodules (44%) and pulmonary hemorrhage (44%). Five patients required ventilation for pulmonary hemorrhage. Four patients (16%) had venous thrombotic events (VTEs). Treatment included prednisone (100%), cyclophosphamide (76%), azathioprine (40%), and methotrexate (32%). CONCLUSION: Pediatric WG typically presents in adolescence and has a female predominance. Glomerulonephritis and pulmonary disease are common at diagnosis and frequently present as a pulmonary-renal syndrome. Loss of renal function is common and rarely completely reversible. As in adults, children with WG are at risk of VTEs.

Atypical and suspicious categories in fine needle aspiration cytology of the breast: histological and mammographical correlation and clinical significance.

INTRODUCTION: This study aims to correlate fine-needle aspiration specimens diagnosed as C3 (atypical, probably benign) and C4 (suspicious, probably malignant) with histology and mammography, and to evaluate these two cytology categories in terms of diagnostic usefulness and patient management. METHODS: All fine-needle aspiration (FNA) specimens in categories C3 or C4 at the Maharaj Nakorn Chiang Mai Hospital, Thailand between 2000-2004 were reviewed. Results were correlated with available histological and mammographical studies. RESULTS: 148 FNA specimens were identified, comprising 43 category C3 and 105 category C4. Histology was available in 90 cases. 14 (64 percent) C3 cases showed benign histology on biopsy and eight (36 percent) were malignant. 13 (19 percent) C4 cases were benign on biopsy, whereas 55 (81 percent) were malignant. Mammographical studies were available in 56 of the histologically-proven cases. All seven cases with benign mammograms had benign histology, and all 26 cases called "highly suggestive of malignancy" were malignant on histology (five C3 and 21 C4). Of the 23 cases called "suspicious abnormality" on mammography, 14 turned out to be malignant on biopsy (one C3 and 13 C4). CONCLUSION: Our study supports maintaining cytology categories C3 and C4. About two-thirds of C3 cases were benign on biopsy whereas 81 percent of C4 cases were malignant (p-value is less than 0.001). There was complete correlation between histological and mammographical studies except those with equivocal mammograms. Our study supports the combined use of clinical, mammographical and cytological findings for optimal patient management. This is especially important for patients with C3 aspiration results, in order to avoid unnecessary surgery for benign lesions.

The inner ear of dogs with X-linked nephritis provides clues to the pathogenesis of hearing loss in X-linked Alport syndrome.

Alport syndrome is an inherited disorder of type IV collagen with progressive nephropathy, ocular abnormalities, and high-tone sensorineural deafness. In X-linked Alport syndrome, mutations in the COL4A5 gene encoding the alpha5 chain of type IV collagen lead to loss of the alpha3/alpha4/alpha5 network and increased susceptibility of the glomerular basement membrane to long-term damage. The molecular defects that underlie the otopathology in this disease remain poorly understood. We used a canine model of X-linked Alport syndrome to determine the expression of type IV collagen alpha-chains in the inner ear. By 1 month in normal adult dogs, the alpha3, alpha4, and alpha5 chains were co-expressed in a thin continuous line extending along the basilar membrane and the internal and external sulci, with the strongest expression along the lateral aspect of the spiral ligament in the basal turn of the cochlea. Affected dogs showed complete absence of the alpha3/alpha4/alpha5 network. The lateral aspect of the spiral ligament is populated by tension fibroblasts that express alpha-smooth muscle actin and nonmuscle myosin and are postulated to generate radial tension on the basilar membrane via the extracellular matrix for reception of high frequency sound. We propose that in Alport syndrome, the loss of the alpha3/alpha4/alpha5 network eventually weakens the interaction of these cells with their extracellular matrix, resulting in reduced tension on the basilar membrane and the inability to respond to high frequency sounds.

Morphological and phenotypic features in pediatric large cell lymphoma and their correlation with ALK expression and the t(2;5)(p23;q35) translocation.

Anaplastic large cell lymphoma (ALCL) was proposed as a clinicopathologic entity over 14 years ago, but has been somewhat controversial due to the variability of its defining features and variable occurrence in different age-groups. To evaluate this entity in a pediatric population, 36 cases of childhood large cell lymphoma were evaluated for abnormalities of the anaplastic lymphoma kinase (ALK) gene that has been associated with ALCL morphology and immunophenotype. ALK abnormalities were evaluated by assay for the t(2;5)(p23;q35) translocation by RT-PCR and/or expression of NPM-ALK fusion protein by immunohistochemistry. Results showed 17 patients to have evidence of ALK gene expression. All of these children (mean age, 9.3 years) had tumors that were of T-cell phenotype (with the exception of a single case of null phenotype) and that expressed CD30. In contrast, 19 children with no evidence of ALK expression were older (mean, 12.7 years), and the majority (12/19) had tumors of B-cell phenotype. CD30 was also diffusely expressed in 8 of these 19 tumors. The difference in mean age between the two groups was statistically significant (P = 0.015). In three cases tested for both ALK and the t(2;5), ALK protein was detected in the absence of the t(2;5) translocation but no cases showed the reverse pattern, consistent with ALK fusion to genes other than NPM or activation of the ALK gene by another mechanism. These findings provide further support that ALK-positive ALCL is a distinct pathologic entity among pediatric large cell lymphomas primarily characterized by expression of T-cell markers, CD30, and EMA, and by a younger mean age.

Regulation of glomerular basement membrane collagen expression by LMX1B contributes to renal disease in nail patella syndrome.

Basement membrane (BM) morphogenesis is critical for normal kidney function. Heterotrimeric type IV collagen, composed of different combinations of six alpha-chains (1-6), is a major matrix component of all BMs (ref. 2). Unlike in other BMs, glomerular BM (GBM) contains primarily the alpha 3(IV) and alpha 4(IV) chains, together with the alpha 5(IV) chain. A poorly understood, coordinated temporal and spatial switch in gene expression from ubiquitously expressed alpha 1(IV) and alpha 2(IV) collagen to the alpha 3(IV), alpha 4(IV) and alpha 5(IV) chains occurs during normal embryogenesis of GBM (ref. 4). Structural abnormalities of type IV collagen have been associated with diverse biological processes including defects in molecular filtration in Alport syndrome, cell differentiation in hereditary leiomyomatosis, and autoimmunity in Goodpasture syndrome; however, the transcriptional and developmental regulation of type IV collagen expression is unknown. Nail patella syndrome (NPS) is caused by mutations in LMX1B, encoding a LIM homeodomain transcription factor. Some patients have nephrosis-associated renal disease characterized by typical ultrastructural abnormalities of GBM (refs. 8,9). In Lmx1b(-/-) mice, expression of both alpha(3)IV and alpha(4)IV collagen is strongly diminished in GBM, whereas that of alpha1, alpha2 and alpha5(IV) collagen is unchanged. Moreover, LMX1B binds specifically to a putative enhancer sequence in intron 1 of both mouse and human COL4A4 and upregulates reporter constructs containing this enhancer-like sequence. These data indicate that LMX1B directly regulates the coordinated expression of alpha 3(IV) and alpha 4(IV) collagen required for normal GBM morphogenesis and that its dysregulation in GBM contributes to the renal pathology and nephrosis in NPS.

Natural history and biology of stage A neuroblastoma: a Pediatric Oncology Group Study.

PURPOSE: To prospectively analyze the outcome of patients with Stage A neuroblastoma (NB) treated with surgery alone, especially with regard to the prognostic significance of age, tumor site, MYCN copy number, tumor cell ploidy, and histology. PATIENTS AND METHODS: The clinical course of 329 patients with Stage A disease registered on the POG NB Biology Study #9047 between February, 1990 and October, 1997 were evaluated. Age, tumor site, MYCN copy number, tumor cell ploidy, and histology were analyzed for their impact on event-free survival (EFS) and survival (S). RESULTS: The 5-year estimated EFS and S rates for the 329 patients were 91% (+/-3%) and 96% (+/-2%), respectively. The EFS rate was similar for infants younger than 12 months and children age 12 months or older, but age older than 12 months was predictive of lower S rates (P = 0.044). Patients with adrenal, abdominal non-adrenal, thoracic, and cervical tumors had similar S rates. The majority of patients had tumors with favorable biologic features, and only 3% had MYCN amplification. For infants with diploid tumors, the EFS rate was 82% (+/-16%), but effective therapy yielded an S rate of 100%. Rate of S was 80% (+/-26%) and 64% (+/-27%) for patients with unfavorable tumor histology and MYCN-amplified tumors, respectively. CONCLUSION: The outcome for patients with Stage A NB treated with surgery alone is excellent. Although EFS and S rates were significantly worse for patients with MYCN-amplified tumors, a subset achieved long-term remission after surgery alone. For patients with Stage A and MYCN amplification, additional factors are needed to distinguish the patients who will achieve long-term remission with surgery alone from those who will develop recurrent disease.

Acute plasmacytic interstitial nephritis in a child with Down syndrome.

A 7.5-year-old boy with Down syndrome presented in acute renal failure (ARF) needing dialysis. When 1.5 years old he had a neuroblastoma, was treated for 1 year with chemotherapy and radiotherapy, and off chemotherapy had since been in remission. Renal biopsy revealed an interstitial inflammation, principally of plasma cells with some lymphocytes and eosinophils. Immunofluorescence showed no deposition of immunoglobulins or complement (C3). The plasma cells were a mixture of kappa and lambda light chain-producing cells. The patient spontaneously improved a week after admission. Initial ultrasonography showed enlarged kidneys with loss of corticomedullary differentiation. We are unaware of a report of ARF in a child, resulting primarily from a polyclonal plasmacytic interstitial nephritis. The etiology remains unclear.

Absence of the alpha6(IV) chain of collagen type IV in Alport syndrome is related to a failure at the protein assembly level and does not result in diffuse leiomyomatosis.

X-linked Alport syndrome is a progressive nephropathy associated with mutations in the COL4A5 gene. The kidney usually lacks the alpha3-alpha6 chains of collagen type IV, although each is coded by a separate gene. The molecular basis for this loss remains unclear. In canine X-linked hereditary nephritis, a model for X-linked Alport syndrome, a COL4A5 mutation results in reduced mRNA levels for the alpha3, alpha4, and alpha5 chains in the kidney, implying a mechanism coordinating the production of these 3 chains. To examine whether production of alpha6 chain is under the same control, we studied smooth muscle cells from this animal model. We determined the canine COL4A5 and COL4A6 genes are separated by 435 bp, with two first exons for COL4A6 separated by 978 bp. These two regions are >/= 78% identical to the human sequences that have promoter activity. Despite this potential basis for coordinated transcription of the COL4A5 and COL4A6 genes, the alpha6 mRNA level remained normal in affected male dog smooth muscle while the alpha5 mRNA level was markedly reduced. However, both alpha5 and alpha6 chains were absent at the protein level. Our results suggest that production of the alpha6 chain is under a control mechanism separate from that coordinating the alpha3-alpha5 chains and that the lack of the alpha6 chain in Alport syndrome is related to a failure at the protein assembly level, raising the possibility that the alpha5 and alpha6 chains are present in the same network. The lack of the alpha6 chain does not obviously result in disease, in particular leiomyomatosis, as is seen in Alport patients with deletions involving the COL4A5 and COL4A6 genes.

Effectiveness of the angiogenesis inhibitor TNP-470 in reducing the growth of human neuroblastoma in nude mice inversely correlates with tumor burden.

Angiogenesis plays an important role in the growth and metastasis of malignant tumors. We have previously reported that in children with neuroblastoma (NB), tumor vascularity directly correlates with metastatic disease, MYCN amplification, and poor outcome. The angiogenesis inhibitor TNP-470 has been shown to reduce the rate of NB growth in rodents with macroscopic tumors without ultimately impacting survival. To investigate whether TNP-470 could more effectively inhibit NB growth in animals with a low tumor burden, we treated 30 nude mice with minimal disease with this angiogenesis inhibitor (supplied by TAP Pharmaceuticals, Inc.). Therapy was initiated before tumors were clinically evident after s.c. inoculation of 5 x 10(6) cells from the MYCN-amplified NB cell line NBL-W-N. TNP-470 was administered 3 days/week, and after 12 weeks of treatment, 53% of the treated mice remained tumor free, whereas 100% of the control mice developed tumors (P < 0.0001). To further assess the relationship between the efficacy of TNP-470 treatment and tumor burden, TNP-470 was also administered s.c., 3 days/week, to mice with clinically evident small (<400 mm3; n = 15) and large (>400 mm3; n = 11) tumors. For animals with small tumors, the mean rate of growth was significantly decreased in the treated mice compared to the controls (P = 0.02). In contrast, there was no difference in the mean rate of tumor growth between animals with large tumors treated with TNP-470 and controls (P = 0.64). Our studies demonstrate that the effectiveness of TNP-470 inversely correlates with tumor burden. We speculate that TNP-470 may most effectively inhibit NB tumor growth in children with a low tumor burden.