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Background: Hydrocephalus is a challenge for pediatric neurosurgeons. When the abdominal cavity and heart fail as diversion sites for cerebrospinal fluid (CSF), many of the otherwise used alternative diversion sites are not feasible due to the smaller physical body size of children and infants. Using the urinary system as a site of diversion has been described in adults primarily.Objective: To describe a minimally invasive procedure to percutaneously access the ureter for placement of a distal catheter in the treatment of pediatric hydrocephalusMethods: A percutaneous ultrasound-assisted technique, was used to access the renal pelvis for catheter placement into the distal ureter.Results: Fifteen months after the surgery the child has a stable neurological condition and adequately managed hydrocephalus.Conclusion: The urinary tract should be considered a viable option for CSF diversion in complex pediatric hydrocephalus. A multidisciplinary approach consisting of interventional radiologists, urologists and neurosurgeons should be involved in the evaluation of potential candidates.

Background Hydrocephalus is a challenge for paediatric neurosurgeons. When the abdominal cavity and heart fail as diversion sites for cerebrospinal fluid (CSF), many of the otherwise used alternative diversion sites are not feasible due to the smaller physical body size of children and infants. Using the urinary system as a site of diversion has been described in adults primarily. Objective To describe a minimally invasive procedure to percutaneously access the ureter for placement of a distal catheter in the treatment of paediatric hydrocephalus. Methods A percutaneous ultrasound-assisted technique was used to access the renal pelvis for catheter placement into the distal ureter. Results Fifteen months after the surgery, the child has a stable neurological condition and adequately managed hydrocephalus. Conclusion The urinary tract should be considered a viable option for CSF diversion in complex paediatric hydrocephalus. A multidisciplinary approach consisting of interventional radiologists, urologists and neurosurgeons should be involved in the evaluation of potential candidates.

Half the children with high-risk neuroblastoma die with widespread metastases. Molecular radiotherapy is an attractive systemic treatment for this relatively radiosensitive tumor.The LuDO-N trial is a phase II, open label, multi-center, single arm, two stage design clinical trial. Children aged 18 months to 18 years are eligible. The trial is conducted by the Nordic Society for Pediatric Hematology and Oncology (NOPHO) and it has been endorsed by SIOPEN (https://www.siopen.net). The Karolinska University Hospital, is the sponsor of the LuDO-N trial, which is conducted in collaboration with Advanced Accelerator Applications, a Novartis company. All Scandinavian countries, Lithuania and the Netherlands participate in the trial and the UK has voiced an interest in joining in 2022.The pediatric use of the Investigational Medicinal Product (IMP)In this paper we present the protocol of the LuDO-N Trial. The rationale and design of the trial are discussed in relation to other ongoing, or planned trials with similar objectives. Further, we discuss the rapid development of targeted radiopharmaceutical therapy and the future perspectives for developing novel therapies for high-risk neuroblastoma and other pediatric solid tumors.

The missense mutation EGFR L858R implies increased sensitivity to EGFR tyrosine kinase inhibitor (TKIs) therapy, despite a significant non-response rate. Currently, detection of EGFR L858R mutation is mostly DNA based, therefore, the allele-specific expression level of the mutated gene and its clinical relevance is hidden. Based on the extendable blocking probes and hot-start protocol for reverse transcription, we have developed and validated a novel one-step realtime RT-PCR assay that enables detection of EGFR L858R mutation at the mRNA level. This RNA-based assay was able to detect the EGFR L858R mutation in a 10,000-fold excess of its wildtype counterpart, indicating an analytical sensitivity of 0.01%. In comparison to the reference DNA-based assay, the RNA-based assay further detected the EGFR L858R mutation in significantly additional formalin-fixed paraffin-embedded (FFPE) samples (19.2% vs 15.0%). Interestingly, our data showed that the relative mRNA levels of EGFR L858R mutation varied greatly in tumor tissues (∼4 logs); and the circulating mRNA of EGFR L858R mutation was detectable in plasma of NSCLC patients. This novel RNA-based PCR assay provides a simple and ultrasensitive tool for detection of EGFR L858R mutation at the mRNA level as a new class of biomarkers.

PURPOSE To evaluate the impact of surgeon-assessed extent of primary tumor resection on local progression and survival in patients in the International Society of Pediatric Oncology Europe Neuroblastoma Group High-Risk Neuroblastoma 1 trial. PATIENTS AND METHODS Patients recruited between 2002 and 2015 with stage 4 disease > 1 year or stage 4/4S with MYCN amplification < 1 year who had completed induction without progression, achieved response criteria for high-dose therapy (HDT), and had no resection before induction were included. Data were collected on the extent of primary tumor excision, severe operative complications, and outcome. RESULTS A total of 1,531 patients were included (median observation time, 6.1 years). Surgeon-assessed extent of resection included complete macroscopic excision (CME) in 1,172 patients (77%) and incomplete macroscopic resection (IME) in 359 (23%). Surgical mortality was 7 (0.46%) of 1,531. Severe operative complications occurred in 142 patients (9.7%), and nephrectomy was performed in 124 (8.8%). Five-year event-free survival (EFS) ± SE (0.40 ± 0.01) and overall survival (OS; 0.45 ± 0.02) were significantly higher with CME compared with IME (5-year EFS, 0.33 ± 0.03; 5-year OS, 0.37 ± 0.03; P < .001 and P = .004). The cumulative incidence of local progression (CILP) was significantly lower after CME (0.17 ± 0.01) compared with IME (0.30 ± 0.02; P < .001). With immunotherapy, outcomes were still superior with CME versus IME (5-year EFS, 0.47 ± 0.02 v 0.39 ± 0.04; P = .038); CILP was 0.14 ± 0.01 after CME and 0.27 ± 0.03 after IME ( P < .002). A hazard ratio of 1.3 for EFS associated with IME compared with CME was observed before and after the introduction of immunotherapy ( P = .030 and P = .038). CONCLUSION In patients with stage 4 high-risk neuroblastoma who have responded to induction therapy, CME of the primary tumor is associated with improved survival and local control after HDT, local radiotherapy (21 Gy), and immunotherapy.

Background There are very few data in the current literature regarding the short- and long-term outcome of surgery for pediatric pancreatic tumors (PPT). No data are available on the impact of pancreatic surgery on the children's growth. Methods This is a retrospective cohort study on a consecutive series of pediatric/adolescent patients who underwent pediatric surgery at Karolinska University Hospital from January 2005 to July 2017. Results Overall 14 pancreatic operations were performed in 13 patients. The median age was 11.4 years (range 3–15). Six pancreaticoduodenectomies (42.8%), 5 distal pancreatectomies (35.7%), and 3 enucleations (21.5%) were performed. The final histology revealed a solid pseudopapillary tumor in 9 cases (69.2%), neuroblastoma in 1 (7.7%), ganglioneuroma in 1 (7.7%), pancreatoblastoma in 1 (7.7%), and insulinoma in 1 (7.7%). Overall, 3 patients developed post-operative complications (23%). There was no peri-operative mortality. All patients are alive after a median follow-up time of 80 months. Exocrine insufficiency was detected post-operatively in 4 patients (30.7%) Endocrine insufficiency requiring insulin treatment developed in one patient (7.7%). No significant impact on growth was detected in any of the patients after pancreatic resection. Conclusions In our series, surgery performed for PPTs seems to be safe and effective. The effect of pancreatic surgery on children's growth does not seem to be significant.

Background The BRAFV600E gene encodes for the mutant BRAFV600E protein, which triggers downstream oncogenic signaling in thyroid cancer. Since most currently available methods have focused on detecting BRAFV600E mutations in tumor DNA, there is limited information about the level of BRAFV600E mRNA in primary tumors of thyroid cancer, and the diagnostic relevance of these RNA mutations is not known. Methods Sixty-two patients with thyroid cancer and non-malignant thyroid disease were included in the study. Armed with an ultrasensitive technique for mRNA-based mutation analysis based on a two step RT-qPCR method, we analysed the expression levels of the mutated BRAFV600E mRNA in formalin-fixed paraffin-embedded samples of thyroid tissues. Sanger sequencing for detection of BRAFV600E DNA was performed in parallel for comparison and normalization of BRAFV600E mRNA expression levels. Results The mRNA-based mutation detection assay enables detection of the BRAFV600E mRNA transcripts in a 10,000-fold excess of wildtype BRAF counterparts. While BRAFV600E mutations could be detected by Sanger sequencing in 13 out of 32 malignant thyroid cancer FFPE tissue samples, the mRNA-based assay detected mutations in additionally 5 cases, improving the detection rate from 40.6 to 56.3%. Furthermore, we observed a surprisingly large, 3-log variability, in the expression level of the BRAFV600E mRNA in FFPE samples of thyroid cancer tissue. Conclusions The expression levels of BRAFV600E mRNA was characterized in the primary tumors of thyroid cancer using an ultrasensitive mRNA-based mutation assay. Our data inspires further studies on the prognostic and diagnostic relevance of the BRAFV600E mRNA levels as a molecular biomarker for the diagnosis and monitoring of various genetic and malignant diseases.

Scrub typhus, caused by Orientia tsutsugamushi, is a common fever in parts of Southern and Southeast Asia. As delayed diagnosis of scrub typhus leads to inappropriate treatment and high mortality rates, of up to 70%, sensitive and rapid detection of O. tsutsugamushi is required for timely and appropriate treatment. Molecular assays, such as PCR and real-time PCR, have been shown to be more sensitive than conventional immunoassay, however, they are only available in centralized laboratories. In contrast to PCR assays, Recombinase Polymerase Amplification (RPA) is conducted under a constant temperature ranging from 24°C to 45°C. Therefore, this technology is very promising for nucleic acid testing in the field, and in resource-limited areas. An RPA assay for the detection of O. tsutsugamushi based on the target gene encoding for the 47 kDa outer membrane protein has been reported, but the primer and probe sequences of this assay are suboptimal for detection of the majority of recently published sequences of O. tsutsugamushi isolates from Southeast Asia. We have established a real-time RPA assay with primer and probe sequences that are optimized for most Southeast Asia's isolates of O. tsutsugamushi. As a result, the new RPA assay showed better performance than the previous assay in detecting O. tsutsugamushi in clinical samples of scrub typhus cases found in Vietnam. The specificity of RPA assay was also evaluated using genomic DNA from microorganisms commonly encountered in the differential diagnosis of scrub typhus, and blood samples from healthy controls and O. tsutsugamushi negative confirmed cases.

The beta subunit of human chorionic gonadotropin (hCG beta) is encoded by six genes (CGB) classified as type I and type II. CGB mRNA is produced in large amounts by trophoblastic tissues and in small amounts by several cancerous tissues including prostate cancer and by a few benign tissues, including the prostate. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) was used to study the expression levels of all CGB mRNAs together (total CGB mRNA) and the two types of CGB mRNA separately in non-cancerous (n = 74) and cancerous prostatic tissue obtained by radical prostatectomy (n = 193). RNA was isolated from formalin-fixed paraffin-embedded (FFPE) samples and mRNA levels of CGB were correlated with disease-specific survival. Total CGB mRNA concentrations were significantly lower (p

Background and Aims: The aim of the study was to clarify the frequency and the sequel of surgical complications occurring within 1 year after renal transplantation. Patients and Methods: Surgical complications after 1670 consecutive adult kidney transplantations performed between 2000 and 2009 were retrospectively analyzed. In 2%, a living-related allograft was used, and 10% were retransplantations. An intravesical technique without stenting was used for the ureteric implantation. Results: There were 282 surgical complications occurring in 259 (15.5%) transplantations. Ureteral obstruction occurred in 53 (3.1%), lymphoceles in 39 (1.5%), postoperative hemorrhage in 36 (2.1%), and renal vein thrombosis in 22 (1.3%) patients, respectively. Out of the 17 lung emboli, 4 were fatal. Male recipients had twice as much ureteral stenosis as female (2.4 vs 1.2%, p Conclusion: Surgical complications impair patient and graft survival after kidney transplantation.

Pancreatic secretory trypsin inhibitor Kazal type 1 (SPINK1) is a 6420 Da peptide produced by the pancreas, but also by several other tissues and many tumors. Some mutations of the SPINK1 gene, like the one causing amino acid change N34S, have been shown to confer susceptibility to recurrent or chronic pancreatitis. Detection of such variants are therefore of clinical utility. So far SPINK1 variants have been determined by DNA techniques. We have developed and validated an immunocapture-liquid chromatography-mass spectrometric (IC-LC-MS) assay for the detection and quantification of serum SPINK1, N34S-SPINK1, and P55S-SPINK1. We compared this method with a time-resolved immunofluorometric assay (TR-IFMA) for serum samples and primer extension analysis of DNA samples. We used serum and DNA samples from patients with acute pancreatitis, renal cell carcinoma, or benign urological conditions. With the help of a zygosity score calculated from the respective peak areas using the formula wild-type (wt) SPINK1/(variant SPINK1 + wt SPINK1), we were able to correctly characterize the heterozygotes and homozygotes from the samples with DNA information. The score was then used to characterize the apparent zygosity of the samples with no DNA characterization. The IC-LC-MS method for SPINK1 was linear over the concentration range 0.5-1000 mu g/L. The limit of quantitation (LOQ) was 0.5 mu g/L. The IC-LC-MS and the TR-IFMA assays showed good correlation. The median zygosity score was 1.00 (95% CI 0.98-1.01, n = 11), 0.55 (95% CI 0.43-0.61, n = 14), and 0.05 (range 0.04-0.07, n = 3) for individuals found to be wt, heterozygous, and homozygous, respectively, for the N34S-SPINK1 variant by DNA analysis. When DNA samples are not available, this assay facilitates identification of the N34S- and P55S-SPINK1 variants also in archival serum samples.

Myotonic dystrophy type 1 (DM1) is an autosomal-dominant disease caused by an expansion of CTG repeats in the 3' untranslated region of the Dystrophia Myotonica Protein Kinase (DMPK) gene. Detection and accurate sizing of the CTG-repeat expansions is clinically important, because the number of CTG repeats correlates with the disease severity. Because difficulties in PCR amplification over large expansions, molecular diagnosis of DM1 is still primarily based on Southern blotting, which is technically demanding and time consuming and requires large amounts of genomic DNA samples. We have recently discovered that the use of multiple heat pulses during Heat Pulse Extension PCR (HPE-PCR) enables efficient amplification over repetitive and GC-rich sequences. Based on this principle, we have developed an assay for efficient amplification of large CTG-repeat expansions seen in DM1 patients. The HPE-PCR method was able to amplify different DMPK1 repeat expansions of up to 1750 CTG repeats in 78 clinical samples with a varying degree of tissue heterogeneity, even in the presence of the short wild-type allele. The CTG-repeat lengths and fragmentation patterns obtained with HPE-PCR were fully concordant with the original diagnostic Southern blotting results. This novel technique provides a PCR-based platform for molecular diagnosis of DM1, and it has been adopted for routine diagnostic use.

Human chorionic gonadotropin (hCG) is a marker of trophoblastic tumors, and the serum concentration of the free beta-subunit (hCGbeta) is an independent prognostic marker in several nontrophoblastic cancers. hCGbeta is encoded by six genes, of which the type II genes (hCGbeta 3/9, 5 and 8) are thought to be upregulated in relation to type I genes (hCGbeta 6/7) in cancer.We developed a novel quantitative RT-PCR method for the quantification of the relative expression levels of the two groups of hCGbeta genes and analyzed 28 bladder tumors and 15 urine samples.We found a higher relative expression level of type II genes in malignant compared with benign urothelia (p = 0.016) and in exfoliated urinary cells from cancer patients compared with those from benign controls (p = 0.026). The expression level was increasing with higher stage (p = 0.014) and grade (p = 0.001) and tended to be higher in relapsing tumors (p = 0.059).The increased hCGbeta concentrations in body fluids of patients with aggressive bladder cancer may be due to overexpression of type II genes. Quantification of the relative mRNA expression levels of the hCGbeta type I and II genes in urine cells should be further studied as a potential noninvasive tool for the diagnosis and follow-up of bladder cancer.

Background: For gene-expression analysis, which is anticipated to play an important role in classification of tumors and premalignant conditions, PCR-based quantitative assays must have increased diagnostic quantitative accuracy and reproducibility and enable analysis of gene expression in formalin-fixed paraffin-embedded (FFPE) tissue samples. Methods: We developed a reverse transcription–PCR-based quantitative assay that modifies the cDNA sequence to increase the melting temperature of short (56–64 bp) PCR amplicons, enabling their quantification in-tube by homogeneous melting-curve analysis. We used this method to analyze the expression of 8 genes, 7 potential colon cancer markers, and 1 control in samples obtained from 3 colon carcinoma cell lines, endoscopic biopsy from 8 patients undergoing gastroscopy for Barrett esophagus, and archival FFPE and frozen tissue from 20 patients who underwent surgery for colon carcinoma. Results: The detection limit of the assay, when optimized for FFPE samples, was 100 copies of cDNA, and the dynamic range was 3 orders of magnitude. A prototype assay containing a panel of 8 genes displayed good reproducibility compared with the commercially available TaqMan® assay (interassay CVs, 5%–20% vs 7%–43%, respectively). Gene-expression analysis was performed successfully in 26 (96%) of 27 endoscopic biopsy specimens, 30 (86%) of 35 archival FFPE samples, and 20 (100%) of 20 archival frozen samples. Conclusions: This new technology combines the reproducibility of competitive PCR with accurate quantitative detection by in-tube melting-curve analysis, enabling efficient analysis of mRNA profiles in samples with small numbers of cells or small amounts of tissue, as well as in archival FFPE tissues.

BACKGROUND. Prostate-specific antigen (PSA/KLK3) and human kallikrein 2 (hK2/KLK2) belong to the human kallikrein gene family. These two highly homologous genes are specifically expressed in the prostate under androgen control. Expression of these is regulated by similar mechanisms but changes in their relative expression have been observed in prostate cancer. METHODS. We determined the relative levels of PSA and hK2 mRNA in benign and malignant prostate tissue using a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. The mRNA of PSA and hK2 are reverse transcribed and amplified in one reaction with the same primers. RESULTS. The variation in the ratio of hK2/PSA mRNA was remarkably small, the difference between the highest and lowest values being three-fold. The ratio was significantly higher in WHO grade 2 compared to normal or benign prostatic hyperplasia tissue (P=0.032 and P=0.035, respectively) and in grade 3 compared to normal or benign prostatic hyperplasia tissue (P=0.006 in both). CONCLUSIONS. The new quantitative RT-PCR technique facilitates very accurate quantitation of the relative mRNA levels of homologous genes. Using this method we have shown that the ratio of hK2/PSA mRNA is higher in cancerous than in benign prostatic tissue. (Less)

Accurate analysis of mRNA expression levels of SNPs, highly homologous genes, and splicing variants requires techniques capable of quantifying low-copy-number mRNAs differing at single nucleotide positions. We have used an RT-PCR-based technique based on co-amplification of closely related target mRNA transcripts and assessed the effect of the stochastic distribution of low-copy-number templates on sampling variation when quantifying rare mRNA transcripts. The technique was optimized for maximal sensitivity to enable the analysis of samples containing a subpopulation of target cells and small microdissected samples. We demonstrate that the input level of template molecules is a critical determinant of the achievable assay precision. A minimum of approximately 50 molecules of template is required to discriminate between 2-fold differences in the expression levels of two transcripts. At levels above 1000 molecules of input template, the stochastic effects on sampling variation become negligible.

Squamous cell carcinoma antigen (SCCA) is widely used as a serum marker in cancers of the uterine cervix, the head and neck, lung and esophagus. Two isoforms of SCCA, deriving from 2 highly homologous serine proteinase inhibitor genes, are co-expressed in normal and malignant squamous epithelium, but it is mainly the acidic isoform SCCA2 that is present in the circulation of cancer patients. We studied the relative levels of SCCA2 and SCCA1 mRNA in frozen sections of squamous cell carcinomas of the head and neck (SCCHN) in relation to disease recurrence, using a new reverse transcription-polymerase chain reaction-based technique for accurate quantitation of relative mRNA levels. Primary tumors from 30 SCCHN patients, recurrent tumors from 11 patients and normal epithelium from 16 controls were examined. In patients responding to initial therapy (n = 26), an elevated SCCA2/SCCA1 mRNA ratio in the primary tumor predicted recurrence independent of clinical stage (p = 0.011). The relative risk of developing a recurrence was 7.2 (CI 1.2-13.3) in patients with elevated vs. normal SCCA2/SCCA1 mRNA ratios. We demonstrate that subtle differences in expression levels of the SCCA genes are reflected in the course of the SCCHN disease and may provide a target for molecular grading of SCCHN tumors. If this finding can be confirmed in a larger study the SCCA2/SCCA1 mRNA ratio in primary tumors could be useful for individual selection of treatment strategy for patients with head and neck cancer.

We studied whether detection of messenger-RNA (mRNA) for the beta-subunit of chorionic gonadotropin (CGbeta) in urinary cells from bladder cancer patients could be used as a marker of disease activity. Sixty-eight urine samples from patients under follow-up for bladder cancer and 23 samples from patients with other malignancies and non-malignant surgical conditions, as well as 14 samples from healthy controls were analyzed. RNA was isolated from urinary cells collected by centrifugation. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect CGbeta mRNA. The results were compared to those obtained by cystoscopy and urinary cytology. For comparison, we determined CG and CGbeta in serum and urine and the core fragment of CGbeta (CGbeta cf) in urine by immunofluorometric assays. CGbeta mRNA was detected in 29 of 68 urine samples from patients with a history of bladder cancer, whereas all 14 samples from healthy controls tested negative. Elevated levels of CGbeta were observed in serum in 18 of 45 bladder cancer patients, but the association with CGbeta mRNA was weak. However, CGbeta mRNA expression in the absence of detectable cancer also occurred in some conditions associated with cellular atypia such as urinary tract infection, instrumentation and certain therapies. There was a highly significant association between histologically verified transitional cell carcinoma of the bladder and CGbeta mRNA in urine (p = 0.0014), implying CGbeta mRNA expression in tumor tissue. We conclude that CGbeta mRNA is a potential new marker for monitoring of bladder cancer. Further studies are needed to evaluate whether it provides independent clinical information.

We used a reverse transcriptase-polymerase chain reaction method for squamous-cell carcinoma (SCC) antigen mRNA to detect circulating tumour cells in patients with carcinoma of the uterine cervix. The sensitivity of the method, as determined by cell spiking experiments, was 10 cultured A431 cells among 10(6) white blood cells. Circulating tumour cells were detected in 6 of 15 patients. In our control group of 24 women, SCC antigen mRNA was detected in 2 pregnant women at term. We followed up the patients for 24 months after sampling and evaluated the outcome. Three out of 6 patients positive for SCC antigen mRNA have relapsed. Additionally, 1 patient has developed breast cancer. In the group of 9 patients negative for SCC antigen mRNA there has been 1 relapse and 1 case of progression of disease. These results suggest that detection of SCC antigen mRNA in peripheral blood by RT-PCR could be useful for staging and evaluation of prognosis in epidermoid carcinoma of the uterine cervix.

Influenza A viruses (IAVs) infect humans and cause significant morbidity and mortality. Different treatment options have been developed; however, these were insufficient during recent IAV outbreaks. Here, we conducted a targeted chemical screen in human nonmalignant cells to validate known and search for novel host-directed antivirals. The screen validated saliphenylhalamide (SaliPhe) and identified two novel anti-IAV agents, obatoclax and gemcitabine. Further experiments demonstrated that Mcl-1 (target of obatoclax) provides a novel host target for IAV treatment. Moreover, we showed that obatoclax and SaliPhe inhibited IAV uptake and gemcitabine suppressed viral RNA transcription and replication. These compounds possess broad spectrum antiviral activity, although their antiviral efficacies were virus-, cell type-, and species-specific. Altogether, our results suggest that phase II obatoclax, investigational SaliPhe, and FDA/EMEA-approved gemcitabine represent potent antiviral agents.

Transverse testicular ectopia (TTE) is a well described, rare congenital abnormality of testicular descent, in which both testes migrate through one inguinal canal. The objective of this work was to present three cases of TTE, one of them with a common vas deferens. To our knowledge, a fused vas deferens has only been reported four times in previously published reports.Three patients presented with inguinal hernia and contralateral cryptorchidism. In case 1, the diagnosis of TTE was made preoperatively by palpating two testes in one hemiscrotum. The diagnosis of case 2 was made intraoperatively and was found to be of a rare form in which the two vasa deferentia fused in the inguinal canal to form a common vas deferens. The diagnosis of case 3 was also done intraoperatively and a laparoscopy was performed to document the anatomy of TTE and to rule out the presence of Müllerian duct remnants. We also performed a literature search for other reports of TTE.The three cases were operated with trans-septal orchidopexy. In addition, laparoscopy was performed in case 3 to clarify the anatomy. Biopsy revealed normal testicular tissue from both testes in the first two patients. Follow-up with ultrasound, 6 months after operation showed normal size and blood flow of both testes.Transverse testicular ectopia should be suspected in a boy with an inguinal hernia and contralateral non palpable testis. Trans-septal orchidopexy is recommended when vasa deferentia are fused. Laparoscopy is useful to document the anatomy and to rule out the presence of Müllerian remnants.

PCR amplification over GC-rich and/or long repetitive sequences is challenging because of thermo-stable structures resulting from incomplete denaturation, reannealing, and self-annealing of target sequences. These structures block the DNA polymerase during the extension step, leading to formation of incomplete extension products and favoring amplification of nonspecific products rather than specific ones. We have introduced multiple heat pulses in the extension step of a PCR cycling protocol to temporarily destabilize such blocking structures, in order to enhance DNA polymerase extension over GC-rich sequences. With this novel type of protocol, we were able to amplify all expansions of CGG repeats in five Fragile X cell lines, as well as extremely GC-rich nonrepetitive segments of the GNAQ and GP1BB genes. The longest Fragile X expansion contained 940 CGG repeats, corresponding to about 2.8 kilo bases of 100% GC content. For the GNAQ and GP1BB genes, different length PCR products in the range of 700 bases to 2 kilobases could be amplified without addition of cosolvents. As this technique improves the balance of amplification efficiencies between GC-rich target sequences of different length, we were able to amplify all of the allelic expansions even in the presence of the unexpanded allele.

Primer-independent cDNA synthesis during reverse transcription hinders quantitative analysis of bidirectional mRNA synthesis in eukaryotes as well as in cells infected with RNA viruses. We report a simple RT-PCR-based assay for strand-specific gene-expression analysis. By modifying the cDNA sequence during reverse transcription, the opposite strands of target sequences can be simultaneously detected by postamplification melting curve analysis and primer-initiated transcripts are readily distinguished from nonspecifically primed cDNA. We have utilized this technique to optimize the specificity of reverse transcription on a panel of 15 target genes. Primer-independent reverse transcription occurred for all target sequences when reverse transcription was performed at 42°C and accounted for 11%–57% of the final PCR amplification products. By raising the reaction temperature to 55°C, the specificity of reverse transcription could be increased without significant loss of sensitivity. We have also demonstrated the utility of this technique for analysis of (+) and (-) RNA synthesis of influenza A virus in infected cells. Thus, this technique represents a powerful tool for analysis of bidirectional RNA synthesis.

Various methods to detect prostatic cells in circulation have given conflicting results. This is probably because qualitative rather than quantitative methods have been used to detect mRNA from prostatic cells. A quantitative method has been developed based on reverse transcription-polymerase chain reaction (RT-PCR) for detection of prostate specific antigen (PSA) mRNA in peripheral blood. A competitive internal mRNA standard was used for quantification of absolute amounts of PSA mRNA. The detection limit of the assay was 7 copies of mRNA, and the highest level of circulating PSA mRNA in 88 control subjects was 25 copies per milliliter of blood. This method was used to study the influence of prostatic surgery and endocrine treatment on prostatic cells in the circulation of 56 patients undergoing biopsy, radical prostatectomy, transurethral resection of the prostate (TURP), orchiectomy, or androgen blockade. Blood samples were drawn before, during and up to 26 weeks after these procedures had been carried out. The highest level of PSA mRNA in controls was 25 copies per milliliter of blood. After RP, TURP or orchiectomy, PSA mRNA levels increased above this level in 27%, 29%, and 25% of the samples, respectively. After prostate biopsy, two out of 15 patients became positive. PSA mRNA levels that were elevated by surgery became undetectable within 1-3 days. No significant correlation was found between PCR positivity and the clinical characteristics of the patients. It is concluded that the level of PSA mRNA in peripheral blood increases after prostatic surgery, indicating temporary dissemination of prostatic cells. However, preoperative levels do not correlate with serum PSA, stage or grade.

ABT-263 and its structural analogues ABT-199 and ABT-737 inhibit B-cell lymphoma 2 (Bcl-2), BCL2L1 long isoform (Bcl-xL) and BCL2L2 (Bcl-w) proteins and promote cancer cell death. Here, we show that at non-cytotoxic concentrations, these small molecules accelerate the deaths of non-cancerous cells infected with influenza A virus (IAV) or other viruses. In particular, we demonstrate that ABT-263 altered Bcl-xL interactions with Bcl-2 antagonist of cell death (Bad), Bcl-2-associated X protein (Bax), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). ABT-263 thereby activated the caspase-9-mediated mitochondria-initiated apoptosis pathway, which, together with the IAV-initiated caspase-8-mediated apoptosis pathway, triggered the deaths of IAV-infected cells. Our results also indicate that Bcl-xL, Bcl-2 and Bcl-w interact with pattern recognition receptors (PRRs) that sense virus constituents to regulate cellular apoptosis. Importantly, premature killing of IAV-infected cells by ABT-263 attenuated the production of key pro-inflammatory and antiviral cytokines. The imbalance in cytokine production was also observed in ABT-263-treated IAV-infected mice, which resulted in an inability of the immune system to clear the virus and eventually lowered the survival rates of infected animals. Thus, the results suggest that the chemical inhibition of Bcl-xL, Bcl-2 and Bcl-w could potentially be hazardous for cancer patients with viral infections.

Quantitation of human chorionic gonadotropin (hCG) in maternal serum is widely used for screening of fetal trisomy-21 (Down's syndrome). When using immunometric assays, the sample usually has to be prediluted in order to quantitate the high hCG concentrations occurring during weeks 10–17 of pregnancy. Utilizing a streptavidin-biotin system we have developed an immunofluorometric assay (IFMA) for the quantitation of hCG in pregnancy serum that does not require predilution of the sample. The sample is added to a streptavidin coated microtiter strip well together with a biotinylated ‘capture’ antibody. About 1–2% of the capture antibody is biotinylated whereas most of the antibodies are unbiotinylated and thus unable to bind to the solid phase, while still binding antigen. This displaces the assay range from 0.5–5000 IU/1 of the standard assay to 20–250 000 IU/1. The method shows good correlation with the standard procedure ( r = 0.96). By eliminating a dilution step the assay procedure is both simplified and reinforced. The principle of this method should be readily applicable to other antigens and detection methods.

The increasing focus on functional genomics spurs new techniques for accurately quantitating differences in mRNA levels.