How is your proctology patient really doing? Outcome measurement in proctology: development, design and validation study of the Proctoprom.

BACKGROUND: The aim of the present study was to develop and evaluate the reliability and validity of proctology patient-reported outcome measurements (PROM): Proctoprom. METHODS: Development of the Proctoprom was based on interview rounds with experts (n = 4) and patients (n = 19) in open informal interview rounds regarding content and form. Once consensus was achieved on five items, data were collected between July 2014 and August 2016 from 991 patients recruited consecutively in a specialized proctology center. Reliability, construct validity and responsiveness of the PROM were determined through exploratory factor analysis, test-retest analysis and anchor-based hypothesis testing. We also estimated discriminant validity, standard error of measurement (SEM), minimal detectable change (MDC95%) and minimal clinically important difference (MCID). RESULTS: The five items loaded on one factor that reflected good internal consistency (Cronbach's α 0.81). Test-retest analysis showed good reliability with intraclass correlation of 0.81. Construct validity measurement resulted in AUCs of 0.85 and 0.90. Responsiveness measurement resulted in AUCs of > 0.76 for both hypotheses. SEM was estimated at 3.0 points and MDC at 4.8 points. We estimated an MCID of 10 points. CONCLUSIONS: Proctoprom is a valid and reliable tool that is responsive to change and that meets consensus-based standards for the selection of health measurement instruments. It can be used to evaluate disease burden and effect of treatment in all adult proctology patients regardless of their proctologic diagnosis.

Perianal fistulas and the lift procedure: results, predictive factors for success, and long-term results with subsequent treatment.

BACKGROUND: Treatment of a perianal fistula is difficult due to the risk of fecal incontinence and recurrence. The ligation of intersphincteric tract (LIFT) procedure is a sphincter-saving procedure associated with success rates ranging from 57 to 94%. The aim of our study was to find predictors for a favorable outcome of the LIFT procedure, evaluation of postoperative fecal incontinence, quality of life, and subsequent treatment with long-term follow-up. METHODS: This study was performed in patients who underwent LIFT between 2013 and 2015 at our institution. Their medical data were retrieved from the electronic patient files. The fistula characteristics were described by physical examination, three-dimensional endoanal ultrasound, and perioperative evaluation. Recurrence rate, postoperative fecal incontinence, and quality of life were assessed with the Patient-Reported Outcome Measurement (PROM). Thirty-two months later, long-term follow-up including subsequent procedures was evaluated. RESULTS: Forty-five patients [17 men, mean age 40 years (range 24-67 years)] were included. In 41 (84%) patients, the fistula was classified as complex; 32 (71%) were referrals with a history of previous fistula surgery. The initial success rate was 18 (40%). Only the height of the internal fistula opening (≥ 15 mm p < 0.03) was associated with recurrence. The LIFT procedure did not affect the occurrence of fecal incontinence or soiling. Recurrence showed a trend with a lower PROM (p = 0.07). Twenty-four months later, further surgery leads to cure in 34 (75%), asymptomatic fistulas in 7 (16%), and persisting active fistulas in 4 (9%) patients. CONCLUSIONS: Initial LIFT had a success rate of 40% and with subsequent surgical treatment 75%. Recurrence after LIFT is related to the height of the internal fistula opening and is associated with diminished quality of life. Continence was not affected by initial LIFT.

Botox treatment in patients with chronic functional anorectal pain: experiences of a tertiary referral proctology clinic.

BACKGROUND: Anorectal pain is a symptom which may have both structural and functional causes, and can, sometimes, develop into a chronic pain syndrome. Functional causes in particular are challenging to treat when conservative treatment measures fail. Botulinum toxin A (BTX-A) can be applied to relax the anal sphincter and/or levator ani muscle to break the vicious circle of pain and contraction. In our tertiary referral proctology clinic, we evaluated the outcome of patients treated with BTX-A for chronic functional anorectal pain. METHODS: Our electronic database was searched for patients who had BTX-A treatment for chronic functional anorectal pain from 2011 to 2016. All medical data concerning history, treatments, and clinical outcome were retrieved. The clinical outcome (resolution of pain) was scored as good, temporary, or poor. RESULTS: A total of 113 patients [47 (42%) males; age 51years, SD 13 years, range 18-88 years] with chronic functional anorectal pain were included. The outcome of BTX-A treatment was good in 53 (47%), temporary in 23 (20%), and poor in 37 (33%). To achieve this outcome, 29 (45%) patients needed a single treatment, 11 (44%) a second treatment, and 13 (54%) ≥ 3 treatments. CONCLUSIONS: Chronic functional anorectal pain can be treated successfully with BTX-A in 47% of patients who fail conservative management. Repeated injections may be needed to ensure complete cure in a subgroup of patients.

Predictive factors for recurrence of cryptoglandular fistulae characterized by preoperative three-dimensional endoanal ultrasound.

AIM: Precise information regarding the location of an anal fistula and its relationship to adjacent structures is necessary for selecting the best surgical strategy. Retrospective and cross-sectional studies were performed to determine predictive factors for recurrence of anal fistula from preoperative examination by three-dimensional endoanal ultrasound (3D-EAUS). METHOD: Patients in our tertiary centre and in a private centre specialized in proctology undergoing preoperative 3D-EAUS for cryptoglandular anal fistulae between 2002 and 2012 were included. A questionnaire was sent in September 2013 to assess the patient's condition with regard to recurrence. Variables checked for association with recurrence were gender, type of centre, previous fistula surgery, secondary track formation and classification of the fistula. RESULTS: There were 143 patients of whom 96 had a low fistula treated by fistulotomy, 28 a high fistula treated by fistulectomy and 19 a high fistula treated by fistulectomy combined with a mucosal advancement flap. The median duration of follow-up was 26 (2-118) months. The fistula recurred in 40 (27%) patients. Independent risk factors included the presence of secondary track formation [hazard ratio 2.4 (95% CI 1.2-51), P = 0.016] and previous fistula surgery [hazard ratio 1.2 (95% CI 1.0-4.6), P = 0.041]. Agreement between the 3D-EAUS examination and the evaluation under anaesthesia regarding the site of the internal opening, classification of the fistula and the presence of secondary tracks was 97%, 98% and 78%. CONCLUSION: The identification of secondary tracks by preoperative 3D-EAUS examination was the strongest independent risk factor for recurrence. This stresses the importance of preoperative 3D-EAUS in mapping the pathological anatomy of the fistula and a thorough search for secondary track formation during surgery.

Linear 2' O-Methyl RNA probes for the visualization of RNA in living cells.

U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Methyl oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.

Synthesis, processing, and transport of RNA within the three-dimensional context of the cell nucleus.

Fluorescence in situ hybridization and immunocytochemical techniques have contributed significantly to our current understanding of how transcription, RNA processing, and RNA transport are spatially and temporally organized in the cell nucleus. New technologies enabling the visualization of nuclear components in living cells specifically advanced our knowledge of the dynamic aspects of these nuclear processes. The picture that emerges from the work reviewed here shows that the positioning of genes within the three-dimensional nuclear space is of crucial importance, not only for its expression, but also for the efficient processing of its transcripts. Splicing factors are recruited from speckles to sites of active transcription, which can be present within, at the periphery, or at a relatively large distance from speckles. Furthermore, results are discussed showing that transcripts are exported by means of random diffusion.

Ribosomal protein genes of yeast contain intervening sequences.

From a colony bank of HindIII-generated yeast DNA fragments we have isolated a number of recombinant DNAs carrying genes for ribosomal proteins (e.g., S10, S16A, S20, S24, S31, S33, L16, L25 and L34) of the yeast Saccharomyces carlsbergensis. By electron microscopic analysis of the R-loops formed between various DNA fragments and yeast mRNA, we could locate the ribosomal protein genes on the physical maps of the cloned DNA fragments. The R-loop structures observed indicate that a number of the ribosomal protein genes contain an intervening sequence.

Alignment of the cell nucleus from labeled proteins only for 4D in vivo imaging.

Studies of protein dynamics by 4D (3D + time) confocal microscopy in vivo are hampered by global cell motion. The time between the acquisitions of the 3D images is in the order of minutes. Therefore, it is not to be expected that the cell as a whole remains fixed in the water basin on the stage. This superimposes a motion on the protein dynamics that has to be removed. We present a robust registration technique to align the cell images that does not require the a priori establishment of point-to-point correspondences. Instead, it uses the distribution of the labeled proteins. After correction for the translation, the 3D rotation of the cell is estimated. A robust intrinsic body coordinate system is constructed via the inertia tensor from the intensity distribution. By combining basis transformation to this intrinsic coordinate system, we can calculated the rotation matrix in a conceptual and computational straightforward manner. We have evaluated the performance of this approach in three experiments with human osteaosarcoma cells (U-2 OS), where the nuclear proteins Histon H4 and PML were visualized. The PML is concentrated in several dozen nuclear spots. Expression of Histon H4 results in a total nuclear staining. The registration results for both channels computed independently are very similar. Practically, this means that only the labeled material needs to be observed and still registration of the cell as a whole can be achieved.

A carboxyl-terminal cysteine residue is required for palmitic acid binding and biological activity of the ras-related yeast YPT1 protein.

The Saccharomyces cerevisiae YPT1 gene codes for a ras-like, guanine nucleotide-binding protein which is essential for cell viability. The functional significance of two consecutive cysteines at the very carboxyl-terminal end of this protein and in ypt homologues of other eukaryotic species was examined. YPT1 gene mutations were generated that either led to substitutions by serine or the deletion of one or both C-terminal cysteines. The consequences of the mutations were checked in cells after replacing the wild type with the mutant genes. It was found that as long as one of the cysteines was retained, the protein was fully functional. The YPT1 protein could be labelled with [3H]palmitic acid that appeared to be bound in an ester linkage. The wild-type protein was evenly distributed between soluble and membrane-associated proteins, the palmitoylated form was predominantly in the crude membrane fraction. The mutant protein lacking the C-terminal cysteines was not palmitoylated and was exclusively found in the soluble fraction. The extension by three residues, -Val-Leu-Ser, generating a ras-typical C-terminal end, did not interfere with the mutant YPT1 protein's function although it resulted in a reduced labelling with palmitic acid.

Methods for visualizing RNA processing and transport pathways in living cells.

Recent advances in fluorescence microscopy, imaging, and probe technology provided possibilities to study the spatial and temporal distribution of RNA species in living cells. While some methods have been developed to localize all nascent or poly (A) containing transcripts others have been developed to study the in vivo distribution of specific RNA species. Irrespective of the method that has been used, the results of these studies provided important information concerning the localization and the cellular transport pathways of RNAs. Also, the picture emerges that RNA molecules travel through the nucleus at much faster speed, equaling that of free diffusion, than previously anticipated. Still, a major challenge proves to be the development of a microscopic detection technique that allows specific, in vivo, detection of low levels of RNA species by fluorescence in situ hybridization, without interfering fluorescent background signals derived from non-hybridized probe sequences and autofluorescent cell components. By applying photoactivatable caged fluorochrome-, molecular beacon-, or fluorescence resonance energy transfer (FRET)-based detection methods an important step in the future of living cell analysis has already been made.

Indications for laparoscopic colorectal surgery. Results from the Medical Centre Alkmaar, The Netherlands.

BACKGROUND: Between November 1991 and May 1995, a series of laparoscopic colectomies were performed in our hospital. METHODS: Our main aim was to define more specifically the indications for laparoscopic colectomy. RESULTS: A total of 69 patients underwent laparoscopic surgery for benign polypoid colorectal disease (n = 10), inflammatory bowel disease (n = 24), and colorectal malignancy (n = 35). Of the latter group, four patients underwent a palliative procedure. The conversion rate of the whole group was 29%. The main reason to convert was infiltrative growth in inflammatory disease or cancer. Respectively, seven (10%) and 12 (17%) patients sustained complications in the perioperative and early postoperative phase. Two patients died perioperatively (3%). The mean hospital stay was 12 days. On follow-up, 11 patients had developed a stenotic anastomosis, which was successfully dilated in all cases. After 3 years, the survival rate according to Kaplan-Meier is 86%, 66%, 68%, and 0% for Dukes' A, B, C, and D color carcinoma, respectively. In one patient with a Dukes B carcinoma, port site metastases were found. CONCLUSIONS: Justifiable indications for laparoscopic colorectal surgery include (a) a benign polyp 20-50 cm from the anal ring; (b) mobile, inflammatory large bowel disease; (c) palliation in case of malignant disease, preferably of the left hemicolon. It remains to be proven that laparoscopic colectomy is superior and not just equivalent to open colectomy. This is especially true for resections of colorectal carcinoma with curative intent. Therefore a cost/benefit analysis should be performed in a prospective, randomized setting.

New insights in the cellular processing of platinum antitumor compounds, using fluorophore-labeled platinum complexes and digital fluorescence microscopy.

The cellular distribution and processing pathways of two platinum compounds, modeling the antitumor drug cisplatin (cDDP) in human osteosarcoma (U2-OS) cells is reported. A [Pt(en)Cl] entity has been covalently linked to a carboxyfluorescein diacetate (CFDA) moiety and to a dinitrophenyl (DNP) moiety. The two different constructs were administered to living cell cultures that were analyzed using digital fluorescence microscopy. The non-fluorescent CFDA construct becomes fluorescent after cellular uptake and subsequent acetate hydrolysis by esterases, and is therefore suitable to monitor platinum in living cells; the DNP construct can be visualized by immunocytochemistry and consequently serves as a control. Both complexes were readily internalized by the cells, and localized throughout the whole cell. After 2-3 h the complex accumulated in the nucleus, but 6-8 h after incubation a punctuate staining of a cytoplasmic region was observed, that persisted and became more pronounced after 24 h. The overall fluorescence in the cell decreased over time, implying a secretion of the platinum complex. Surprisingly, the accumulation remained visible after 72 h. Co-localization experiments with a Golgi apparatus-selective stain indicate the involvement of Golgi vesicles in intracellular processing of cisplatin-derived complexes. Immunocytochemical studies, using the DNP derivative, resulted in very similar images as obtained with the CFDA construct. CFDA-boc (a non-platinum-containing fluorescein derivative) was used as control: a faint staining throughout the whole cell was observed. Cisplatin-resistant U2-OS/Pt cells showed staining patterns very similar to the U2-OS cells using both platinum constructs. This study illustrates that only a very small portion of the platinum complex eventually remains bound to DNA, as after 24 h no significant fluorescence could be observed in the nucleus. Cisplatin-derived complexes with fluorescent tags afford a new insight into the cellular processing of these complexes and therefore may contribute to further unraveling of the mechanism of platinum antitumor complexes.

The primary structure of a gene encoding yeast ribosomal protein L34.

Sequence analysis revealed that a gene coding for yeast ribosomal protein L34 comprises an amino acid coding region of 339 nucleotides which is interrupted by an intron after the 19th codon. Like for other yeast ribosomal protein genes analyzed thus far a strong codon bias was observed. The flanking and intervening sequences of this gene encoding L34 show several elements that are conserved in a number of split ribosomal protein genes in yeast. Northern blot analysis using an intron-specific probe demonstrated that the sequenced gene copy coding for L34 is transcribed in vivo.

Biochemical properties of the ras-related YPT protein in yeast: a mutational analysis.

Using site-directed mutagenesis, the ras-related and essential yeast YPT1 gene was changed to generate proteins with amino acid exchanges within conserved regions. Bacterially produced wild-type proteins were used for biochemical studies in vitro and were found to have properties very similar to mammalian ras proteins. Gene replacement allowed the study of physiological consequences of the mutations in yeast cells. Lys21----Met and Asn121----Ile substitutions rendered the protein incapable of binding GTP and caused lethality. Ser17----Gly and Ala65----Thr substitutions slightly changed the protein's apparent binding capacity for either GDP or GTP and altered its intrinsic GTPase activity. These mutations were without effect on cellular growth. The YPTgly17,thr65 mutant protein displayed a significantly altered relative capacity for guanine nucleotide binding but a GTPase activity comparable to the wild-type protein. In contrast to the Ala65----Thr substitution, the double mutant displayed a significantly reduced capacity for autophosphorylation and allowed cells to grow only poorly. Cellular growth was improved when this mutant protein was overproduced.

Molecular cloning and characterization of mouse CD97.

The EGF-TM7 family (CD97 and EMR1) is a group of class II seven-span transmembrane receptors predominantly expressed by cells of the immune system. Recently, we have identified CD55, a regulatory molecule of the complement cascade, as a cellular ligand of human CD97 (hCD97). In this study, the molecular properties of mouse CD97 (mCD97) are described. Like hCD97, mCD97 has an extended extracellular region with several epidermal growth factor-like (EGF) domains. Due to alternative RNA splicing, isoforms with three and four EGF domains exist, designated mCD97(EGF1,2,4) and mCD97(EGF1,2, 3,4) respectively. All EGF domains, except for the N-terminal one, possess a calcium-binding site. In a third isoform mCD97(EGF1,2,X,3, 4), a sequence of 45 amino acids was found between the second and third EGF domain that does not correspond to any known protein module. Using newly generated mCD97 mAb, we show that analogous to the blood expression pattern of hCD97, mCD97 can be found on lymphoid and myeloid cells. Adhesion of mouse erythrocytes and splenocytes to COS cells expressing mCD97(EGF1,2,4) or mCD97(EGF1,2, 3,4) could be blocked by mouse CD55 (mCD55) antibody, identifying mCD55 as a cellular ligand for mCD97. Consistent with the necessity of directly linked EGF domains for the integrity of the CD55-binding site on hCD97, no adhesion was detected to the largest mouse isoform mCD97(EGF1,2,X,3,4). Remarkably, we found that the interaction between CD97 and CD55 is phylogenetically restricted, as indicated by the selective adhesion of primate erythrocytes to hCD97 transfectants, and of mouse and rat erythrocytes to mCD97 transfectants respectively.

The structure of the gene coding for the phosphorylated ribosomal protein S10 in yeast.

From previous studies on cloned yeast ribosomal protein genes we obtained evidence that a large number of them contain an intron [Bollen et al. (1982) Gene 18, 29-38]. In the temperature-sensitive rna2-mutant transcription of these genes leads to the accumulation of precursor RNAs at the restrictive temperature. These precursor mRNAs are several hundreds of nucleotides longer than the respective mature mRNAs. The split character of one of these ribosomal protein genes, viz. the gene coding for the major phosphorylated small-subunit protein S10, was further established by sequence analysis. The intervening sequence interrupts the coding sequence after the second codon and has a length of 352 nucleotides. Genomic Southern hybridizations with a DNA fragment carrying part of the S10-gene revealed that this gene is duplicated on the yeast genome. The molecular weight of S10 as deduced from the sequence analysis was estimated to be 31462 dal. Comparison of the N-terminal aminoacid sequence of the yeast ribosomal protein S10 with that of ribosomal protein S6 from rat liver revealed a striking homology between both proteins. Moreover, at the C-terminal end of the yeast ribosomal protein the sequence Arg-Ala-Ser-Ser-Leu-Lys is present which is very similar to the phosphorylation site of the rat liver protein S6.

Appearance of IgG and IgA antibodies in human bile after tetanus toxoid immunization.

Humans immunized intramuscularly with one dose of tetanus toxoid exhibited IgG, and in some cases IgA antibody, in their bile as well as serum. Both isotypes appeared in bile transiently with titres declining after about day 10 for both classes. These kinetics resembled those of the serum IgA response but were markedly different to those for IgG antibody in serum. Measured IgG titres in bile were between 0.07 and 4.2% of those in paired sera, and IgA titres were between 6.8 and 124% of sera. Peak responses in bile, while generally of smaller size, exceeded those of paired sera when expressed as antibody/mg of IgG or IgA present. This calculation showed that during the peak response bile was up to nineteen-fold more abundant in IgG antibody than was serum taken at the same time, and up to forty-five-fold more for IgA. Enrichment of antibody in bile is not consistent with the Ig of bile being solely conferred by plasma, and may mean the involvement of local synthesis too. This study indicates that tetanus toxoid immunization of humans results in biliary antibody and raises the possibility of intra-hepatic antibody production for export to the intestinal tract in man.

Yeast contains two functional genes coding for ribosomal protein S10.

The DNA sequence of the second copy of the gene coding for yeast ribosomal protein S10 was determined and compared with the sequence of the first gene-copy. In addition, the sites at which the transcription of these genes start and terminate are identified. The amino acid coding regions of the two gene copies are virtually identical. The leader and in particular the trailer sequences, however, are significantly different, while the intervening sequences have hardly any homology. Taking advantage of the sequence differences we could establish that both genes are expressed in the vegetatively growing yeast cell; the respective transcripts, however, differ in their relative amounts.