Standardization of catheter load speed during embryo transfer: comparison of manual and pump-regulated embryo transfer.

The position of transfer air bubbles after embryo transfer is related to the pregnancy rate. With the conventional manual embryo-transfer technique it is not possible to predict the final position of the air bubbles. This position mainly depends on the catheter load speed at transfer (injection speed), a parameter that remains uncontrollable with the conventional technique even after standardization of the protocol. Therefore, the development of an automated device that generates a standardized injection speed is desirable. This study aimed to examine the variation in injection speeds in manual embryo transfer and pump-regulated embryo transfer (PRET). Seven laboratory technicians were asked to perform simulated transfers using the conventional embryo-transfer technique. Their injection speeds were compared with that of a PRET device. The results indicate that in manually performed transfers, even after standardization of the protocol, there is still a large variation in injection speed, while a PRET device generates a reliable and reproducible injection speed and therefore brings new possibilities for further standardization of the embryo-transfer procedure. Future research should reveal whether these experiments mimic real clinical circumstances and if a standardized injection speed results in more exact positioning of the transferred embryos and therefore higher pregnancy rates.

Human adenovirus type 35 vector for gene therapy of brain cancer: improved transduction and bypass of pre-existing anti-vector immunity in cancer patients.

Clinical trials in malignant glioma have demonstrated excellent safety of recombinant adenovirus type 5 (Ad5) but lack of convincing efficacy. The overall low expression levels of the Coxsackie and Adenovirus receptor and the presence of high anti-Ad5-neutralizing antibody (NAb) titers in the human population are considered detrimental for consistency of clinical results. To identify an adenoviral vector better suited to infect primary glioma cells, we tested a library of fiber-chimeric Ad5-based adenoviral vectors on 12 fresh human glioma cell suspensions. Significantly improved marker gene expression was obtained with several Ad5-chimeric vectors, predominantly vectors carrying fiber molecules derived from B-group viruses (Ad11, Ad16, Ad35 and Ad50). We next tested Ad35 sero prevalence in sera derived from 90 Dutch cancer patients including 30 glioma patients and investigated the transduction efficiency of this vector in glioma cell suspensions. Our results demonstrate that the sero prevalence and the titers of NAb against Ad35 are significantly lower than against Ad5. Also, recombinant Ad35 has significantly increased ability to transfer a gene to primary glioma cells compared to Ad5. We thus conclude that Ad35 represents an interesting candidate vector for gene therapy of malignant glioma.

Isolation and characterization of the mouse homolog of SYT, a gene implicated in the development of human synovial sarcomas.

In a previous study we reported the isolation of the human synovial sarcoma-associated t(X;18) breakpoint. As a result of this translocation, the SYT gene on chromosome 18 fuses to either the SSX1 or the SSX2 gene on the X chromosome, depending on the exact location of the breakpoint within band Xp11.2. As yet, little is known about the modes of action of the SYT and SSX genes and their respective (fusion) products. Here we report the isolation of the mouse homolog of SYT, its full length cDNA sequence, its chromosomal localization, and its spatio-temporal expression patterns in adult and embryonic tissues. The SYT gene was found to be well conserved during evolution and is part of a region of synteny between the human and mouse chromosomes 18. In early embryogenesis, Syt is ubiquitously expressed. In later stages, the expression becomes confined to cartilage tissues, specific neuronal cells and some epithelial derived tissues. In mature testis, expression was specifically observed in primary spermatocytes.

Sublocalization of the synovial sarcoma-associated t(X;18) chromosomal breakpoint in Xp11.2 using cosmid cloning and fluorescence in situ hybridization.

In a previous study we localized the synovial sarcoma-associated t(X;18)(p11;q11) breakpoint within the ornithine aminotransferase-like 1 (OATL1) cluster on the X chromosome. This localization was delineated from both somatic cell hybrid and fluorescence in situ hybridization (FISH) analysis of patient material, using OAT-specific cDNA and YAC probes. Simultaneously, Knight et al. (1992, Mol. Hum. Genet, in press) mapped this same breakpoint in their patient material adjacent to the more proximal OATL2 region on the X chromosome. Here we report the analysis of two additional tumors and demonstrate that again in these cases the chromosomal break occurs within the OATL1 cluster. In order to further specify the breakpoint, we subcloned the OATL1 YAC (no. 2) into cosmids. At least one of these cosmids (0.38) hybridizes to sequences that bracket the translocation breakpoint, as demonstrated by both Southern blot and FISH analysis. These observations confirm and substantiate our previous findings. In addition, cosmid 0.38 should be a valuable instrument for the ultimate isolation and identification of the gene(s) involved in the development of synovial sarcoma.

Distinct Xp11.2 breakpoint regions in synovial sarcoma revealed by metaphase and interphase FISH: relationship to histologic subtypes.

Fluorescence in situ hybridization (FISH) and molecular analyses of synovial sarcomas with cytogenetically similar (X;18)(p11.2;q11.2) translocations have revealed two alternative breakpoint regions in Xp11.2, one residing in the ornithine aminotransferase-like 1 (OATL1) region and the other one in the related but distinct OATL2 region. As these results were obtained by different groups, we set out to evaluate an extended series of tumors with special emphasis on the two possible X-related breakpoint regions. Together, seven synovial sarcomas were identified with a break in the OATL1 region and six with a break near OATL2, thereby confirming the actual existence of the two alternative Xp breakpoint regions. We speculate that there seems to be a relationship between the occurrence of these breakpoint regions and the histologic phenotype of the tumors, with a predominance of OATL1-related breakpoints in the classical biphasic tumors and of OATL2-related breakpoints in the monophasic fibrous tumors.

Identification of a yeast artificial chromosome that spans the human papillary renal cell carcinoma-associated t(X;1) breakpoint in Xp11.2.

Recently, a specific chromosome abnormality, t(X;1)(p11;q21), was described for a subgroup of human papillary renal cell carcinomas. The translocation breakpoint in Xp11 is located in the same region as that in t(X;18)(p11;q11)-positive synovial sarcoma. We used fluorescence in situ hybridization (FISH) and somatic cell hybridization techniques to demonstrate 1) that the Xp11 translocation breakpoint in papillary renal cell carcinoma differs from that observed in synovial sarcoma and has a more proximal location, and 2) that an ornithine aminotransferase (OAT)L2 containing yeast artificial chromosome (YAC) spans the X;1 translocation. This YAC provides an ideal starting point from which the breakpoint itself and the gene(s) involved can be isolated and characterized.

Comparison of the effect of different chitosan salts and N-trimethyl chitosan chloride on the permeability of intestinal epithelial cells (Caco-2).

A partially quaternized chitosan derivative, N-trimethyl chitosan chloride (TMC) (degree of quaternization 12.28%), was synthesized and the effects of this novel polymer on the permeability of intestinal epithelial cells, using Caco-2 cell monolayers, were investigated and compared with those of chitosan hydrochloride and chitosan glutamate. Transepithelial electrical resistance (TEER) measurements at pH 6.20 revealed that all these polymers (0.25-1.5% w/v) caused an immediate and pronounced lowering in TEER values in the order chitosan hydrochloride (84% reduction after 2 h incubation) > chitosan glutamate (60% reduction) > TMC (24% reduction) at 0.25% w/v concentrations. At higher concentrations (up to 2.5% w/v), TMC was able to decrease the TEER further. Similar results were obtained in transport studies, using the hydrophilic radioactive markers, [14C]-mannitol (MW 182.2) and [14C]-polyethylene glycol 4000 (PEG-4000, MW 4000). Large increases in the permeation of these markers were found. The transport of [14C]-mannitol was increased 34-fold (chitosan hydrochloride), 25-fold (chitosan glutamate) and 11-fold (TMC) at 0.25% w/v concentrations. Further increases in the permeation of both markers were found at higher concentrations of TMC. Due to its quaternary structure, TMC is better soluble than the other chitosan salts, and its higher solubility may compensate for its lesser effectivity at similar concentrations. It is also soluble at pH 7.40, where the chitosan salts are insoluble and therefore ineffective. No deleterious effects to the cells could be demonstrated with trypan blue exclusion studies and confocal laser scanning microscopy (CLSM). CLSM confirmed that these polymers increase the transport of large hydrophilic compounds (using the fluorescent markers FD-4, MW 4400 and FD-20, MW 19,600) through opening of tight junctions to allow for paracellular transport. It is concluded from this study that the charge, charge density and the structural features of chitosans and chitosan derivatives are important factors determining their potential use as absorption enhancers.

Severe extrapyramidal syndrome in a dog caused by a Haloperidol (Serenase®) intoxication.

Summary The case history of a one-year-old male mongrel dog intoxicated with 120 mg haloperidol is described. The dog showed a coma with a severe extrapyramidal syndrome and was treated with orphenadrine. Symptoms, occurrence, and therapy of the extrapyramidal syndrome are discussed. Emphasis is laid on the importance to differentiate this syndrome from epilepsy and other neurological disorders.

Dentistry's role in obstructive sleep apnoea. Review and case report.

Obstructive sleep apnoea (OSA) has been associated with many life-threatening conditions but has only recently appeared in the dental literature. Dental appliances and orthognathic surgery are two strategies which are currently used in the treatment of sleep apnoea. This article provides a background on OSA and these treatment approaches, and discusses the potential risks and benefits of each. A case is reported to illustrate the use of a dental appliance in the treatment of OSA.

[Immunology in the clinical practice. XVI. Paraneoplastic syndromes of the nervous system: pathogenesis and diagnosis]. / Immunologie in de medische praktijk. XVI. Paraneoplastische syndromen van het zenuwstelsel: pathogenese en diagnostiek.

Paraneoplastic neurological syndromes are believed to result from ectopic expression of onconeural antigens by tumours. The resulting immune response is not only directed against the tumour but also cross-reacts with the same or similar antigens in the nervous system. The immune response generates high titred autoantibodies that are associated with specific tumours and neurological syndromes. Paraneoplastic autoantibodies help diagnose neurological syndromes and help direct the search for an underlying tumour. In paraneoplastic syndromes, the course of the tumour is relatively mild. Detection of the autoantibodies might lead to early diagnosis and immunomodulation and anti-tumour treatment before irreversible neuronal cell loss and deficits set in.

Severe extrapyramidal syndrome in a dog caused by a haloperidol (Serenase) intoxication.

The case history of a one-year-old male mongrel dog intoxicated with 120 mg haloperidol is described. The dog showed a coma with a severe extrapyramidal syndrome and was treated with orphenadrine. Symptoms, occurrence, and therapy of the extrapyrmidal syndrome are discussed. Emphasis is laid on the importance to differentiate this syndrome from epilepsy and other neurological disorders.

[Tissue antigens and bacterial infection (author's transl)]. / Weefselantigenen en microbiële infecties.

Rejection of tissue- and organ-grafts is due to the existence of tissue- or transplantation-antigens which are present in principle on every nucleated body-cell. These tissue antigens represent the phenotypical expression of a complex system of genes which are localized on an autosomal chromosome: the histocompatibility system. These systems may usually be compared with bloodgroup systems. Besides the successful use of tissue typing in transplantation, it became apparent that histocompatibility systems may have another function. Among others, tissue typing may be correlated with resistance to and susceptibility to particular diseases. However, it is not clear so far whether tissue antigens play a role themselves or only serve as genetic markers in this regulation of the immune response. The two possibilities are introduced, together with some instances. Finally, on the basis of possible veterinary and economic importance, studies on histocompatibility systems in pets and farm animals are reviewed, a number of possible uses being suggested.

FOXL2 and SOX9 as parameters of female and male gonadal differentiation in patients with various forms of disorders of sex development (DSD).

The transcription factors SOX9 and FOXL2 are required for male and female mammalian gonadal development. We have used specific antibodies to investigate the role of these key proteins in disorders of sex development (DSD), specifically inter-sex states. In normal gonads, SOX9 was found to be restricted to the presence of (pre-)Sertoli cells, while FOXL2 was found in granulosa cells, and in stromal cells interpreted as early ovarian stroma. Both proteins were found within a single patient, when testicular and ovarian development was present; and within the same gonad, when both differentiation lineages were identified, as in ovotesticular DSD (ie hermaphrodite). Especially SOX9 was informative to support the presence of early testicular development (ie seminiferous tubules), expected based on morphological criteria only. In a limited number of DSD cases, FOXL2 was found within reasonably well-developed seminiferous tubules, but double staining demonstrated that it was never strongly co-expressed with SOX9 in the same cell. All seminiferous tubules containing carcinoma in situ (CIS), the malignant counterpart of a primordial germ cell, ie the precursor of type II germ cell tumours of the testis, seminomas and non-seminomas, showed the presence of SOX9 and not FOXL2. In contrast, gonadoblastomas (GBs), the precursor of the same type of cancer, in a dysgenetic gonad, showed expression of FOXL2 and no, or only very low, SOX9 expression. These findings indicate that gonadal differentiation, ie testicular or ovarian, determines the morphology of the precursor of type II germ cell tumours, CIS or GB, respectively. We show that in DSD patients, the formation of either ovarian or/and testicular development can be visualized using FOXL2 and SOX9 expression, respectively. In addition, it initiates a novel way to study the role of the supportive cells in the development of either CIS or GB.

A novel Krüppel-associated box containing the SSX gene (SSX3) on the human X chromosome is not implicated in t(X;18)-positive synovial sarcomas.

The human synovial sarcoma-specific translocation t(X;18) results in the fusion of the SYT gene on chromosome 18 with either one of the Krüppel-associated box (KRAB) containing SSX1 or SSX2 genes on the X chromosome, depending on the exact location of the breakpoint within band Xp11.2. Screening of a testis cDNA library yielded several SSX-positive clones. Subsequent sequence analysis revealed that one third of these clones represent an SSX gene that differs from both SSX1 and SSX2. This novel member of the family of KRAB containing SSX genes, which we designated SSX3, is 90% homologous to SSX1 and 95% homologous to SSX2 at the cDNA level. Somatic cell hybrid analysis indicated that SSX3 maps within Xp11.2 --> p11.1, the region that also harbors the SSX1 and SSX2 genes. However, we conclude from our RT-PCR data and from results reported in the literature that SSX3 does not act as a fusion partner to SYT in any of the 44 independent synovial sarcomas thus far tested.

A 5.5-Mb high-resolution integrated map of distal 11q13.

The distal part of 11q13, which contains several genes relevant to human diseases, has been poorly mapped as part of genome-wide mapping efforts. In the prospect of drawing a fine-scale integrated map of the area containing KRN1 and OMP, we have established a framework of markers by hybridization to DNA of somatic cell hybrids and by fluorescence in situ hybridization (FISH) on metaphase chromosomes. The probes studied were used to isolate 27 YACs and 16 cosmids that could be organized in three contigs covering approximately 6 Mb. These contigs were separated by two gaps that are likely to contain sequences underrepresented in YAC libraries. They were then integrated based on long-range restriction mapping and DNA-fiber FISH into a high-resolution physical map, which covers a 5.5-Mb region and includes 36 anonymous markers and 10 genes. This map will be used to search for genes within the 2/3 of this region where none have been localized as yet. It will also lay the ground for the characterization of an amplicon surrounding GARP in breast cancer and for the search of disease genes within this region.

Mucoadhesive polymers in peroral peptide drug delivery. VI. Carbomer and chitosan improve the intestinal absorption of the peptide drug buserelin in vivo.

PURPOSE: To evaluate the effect of the crosslinked poly(acrylate) carbomer 934P (C934P) and its freeze-dried neutralized sodium salt (FNaC934P) as well as chitosan hydrochloride on the intestinal absorption of the peptide drug buserelin. METHODS: Buserelin was applied intraduodenally in control buffer, 0.5% (w/v) C934P, 0.5% (w/v) FNaC934P, 1.5% (w/v) chitosan hydrochloride or FNaC934P/chitosan hydrochloride (1:1 (v/v)) mixture in rats. RESULTS: All polymer preparation showed a statistically significant improvement of buserelin absorption compared to the control solution. The absolute bioavailabilities for the different polymer preparations were: control, 0.1%; 0.5% FNaC934P, 0.6%; 0.5% C934P, 2.0%; chitosan hydrochloride, 5.1% and FNaC934P/chitosan hydrochloride (1:1 (v/v)) mixture, 1.0%. The higher bioavailability with chitosan hydrochloride compared to C934P and FNaC934P indicates that for buserelin the intestinal transmucosal transport enhancing effect of the polymer plays a more dominant role than the protection against proteases such as alpha-chymotrypsin. CONCLUSIONS: The mucoadhesive polymers carbomer 934P and chitosan hydrochloride are able to enhance the intestinal absorption of buserelin in vivo in rats, and may therefore be promising excipients in peroral delivery systems for peptide drugs.