Comparative studies on the amino acid derivatives of indometacin

A series of amino acid derivatives of indometacin (IND) was investigated in regard to their protein binding and prostaglandin synthetase inhibition in vitro, and to acute toxicity, anti-inflammatory, antiedemic, analgesic actions, and the influence on the central nervous system in vivo. In biochemical tests the compounds were several times less potent than IND. They differed among themselves in the respect of toxicity, which was always much lower than that of IND. Out of eight compounds investigated N-IND-glycine (K1) and N-IND-epsilon-aminocaproic acid (K5) exerted more favorable antiedemic and analgesic action than IND did. Both the derivatives only weakly inhibited the cotton-pellet granuloma formation. K1 acted similarly to IND in the arthritis test. K1, K5 and IND similarly irritated the gastric mucosa. A modification of IND structure by introduction of glycine or epsilon-aminocaproic acid resulted in two new anti-inflammatory agents of more favorable therapeutic index in the antiedemic and analgesic action and of much lower toxicity than the reference compound.     

An open-label efficacy pilot study with pimecrolimus cream 1% in adults with facial seborrhoeic dermatitis infected with HIV

BACKGROUND: Seborrhoeic dermatitis (SD) is a common dermatosis in human immunodeficiency virus (HIV)-positive patients, many of whom do not respond satisfactorily to conventional topical treatments such as corticosteroids and antifungals. OBJECTIVE: A pilot study to investigate the efficacy and tolerability of pimecrolimus cream 1% in HIV-positive patients with facial SD. METHODS: In a single-centre study, 21 HIV-infected patients with mild to severe SD were treated twice daily with pimecrolimus cream 1% for 14 days. Thereafter, treatment was discontinued and patients followed up for 5 weeks. Skin involvement at baseline and on days 7, 14, 21, 35 and 49 was assessed using a four-point clinical score and digital photography. MAIN OUTCOME MEASURES: Efficacy and safety of pimecrolimus cream 1% treatment and incidence of relapse in the follow-up phase. Results Marked improvement was seen in clinical parameters at day 7, with >or= 90% patients clear of symptoms at day 14. Relapse was observed at day 35 but signs were milder than at baseline. All patients responded to therapy, despite their immunological status. Pimecrolimus did not alter CD4(+) and CD8(+) T-cell counts or viral load during the treatment period. CONCLUSION: Pimecrolimus cream represents a new, effective therapeutic option for facial SD in HIV patients.     

In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium

In-vitro maturation (IVM) of oocytes is a promising technique to reduce the costs and avert the side-effects of gonadotrophin stimulation for in-vitro fertilization (IVF). The pregnancy rates from oocytes matured in vitro are much lower than those of in-vivo stimulation cycles indicating that optimization of IVM remains a challenge. Therefore, we investigated the effect of supplementation of the medium with gonadotrophins, oestradiol and epidermal growth factor (EGF) and the effect of retaining or removing the cumulus cells on nuclear and cytoplasmic maturation of immature oocytes. Human germinal vesicle (GV) oocytes obtained after gonadotrophin stimulation for intracytoplasmic sperm injection (ICSI) were cultured in a complex defined medium either supplemented with gonadotrophins, oestradiol and physiological concentrations of EGF (2 ng/ml) or gonadotrophins and oestradiol alone. The cumulus cells were either removed or kept intact. In GV stage oocytes cultured without cumulus (group I) significantly more oocytes reached the metaphase II (MII) stage at 30 h in media supplemented with EGF (64.3 versus 33.9%, P < 0.003). For oocytes cultured with intact cumulus (group II), more oocytes reached MII at 30 h than in group I, but there was no difference in medium with or without EGF supplementation (81.8 and 79.8% respectively). Cytoplasmic maturation of MII oocytes was judged from their capability to activate and fertilize after ICSI. In group I, the rates of activation and normal fertilization were similar. However, in group II, significantly more oocytes underwent normal fertilization in the EGF-supplemented than the unsupplemented group (71.7 versus 45.6%, P < 0.05). The cleavage rates of the fertilized oocytes were similar in the sibling oocyte subgroups cultured with or without EGF supplementation, but the overall cleavage rates were higher in cumulus-intact compared to cumulus-denuded oocytes (88.9 versus 47.8%, P < 0.001). Thus, supplementation of the maturation medium with EGF and maintenance of the cumulus during culture improve the nuclear and cytoplasmic maturation of human oocytes in vitro.      

The effects of co-culture with human fibroblasts on human embryo development in vitro and implantation

In a human in-vitro fertilization (IVF) programme, the effect of co- culture of embryos with human fibroblasts was evaluated with respect to pregnancy rate and embryo development. Patients were included in the study after giving informed written consent. The IVF treatments were randomly assigned by stratification of both age (<36 versus > or =36 years) and previous IVF attempts (yes versus no). After fertilization was established, the zygotes were transferred to a 4-well dish with or without fibroblasts and cultured for 2 days. On the third day after ovum pick-up (OPU), cell number and quality [5 (good) to 1 (poor)] of the embryos were scored and a maximum of three embryos was transferred. Supernumerary embryos of good quality were cryopreserved. The design of this study was a group sequential trial with the objective of detecting differences between pregnancy rates following IVF with conventional incubation or incubation in co-culture with fibroblasts. This design included one evaluation at half-way data collection. In the study, 148 patients had an OPU, of whom 77 were allocated to the co-culture group. There was no statistically significant difference in pregnancy rate, cell number and embryo quality between the two groups. The ongoing pregnancy rate per embryo transfer was 27% in co-culture and 30% in the conventional culture group. The implantation rates per transferred embryo were 17 and 18% respectively. Using a multivariate logistic regression model for the probability of ongoing pregnancies, the odds ratio of co-culture, adjusted for age and previous IVF attempts, was not statistically significant. In conclusion, co-culture with human fibroblasts does not contribute to an improvement of embryo quality nor to a higher pregnancy rate after IVF in an unselected group of patients.      

Breast carcinoma diffusely metastatic to the spleen. A report of two cases presenting as idiopathic thrombocytopenic purpura.

Carcinoma metastatic to the spleen is found at autopsy in 6% to 13% of patients who die of cancer, yet clinical symptoms referable to splenic metastases are unusual. Two cases of breast carcinoma metastatic to the spleen discovered incidentally at therapeutic splenectomy for idiopathic thrombocytopenic purpura are described. On gross examination, the spleens were mildly enlarged with a homogeneous congested cut surface; rare 0.2-cm white nodules were present in one case. Microscopic examination revealed large, poorly cohesive cells that diffusely involved both the red and white pulp. Histochemical, immunohistochemical, and ultrastructural analyses confirmed the epithelial nature of the cellular infiltrate. These cases show that idiopathic thrombocytopenic purpura may herald the presence of diffuse splenic metastases when metastatic disease is not otherwise clinically suspected. The lack of a discrete tumor mass in the spleen in such cases may make the diagnosis of metastatic carcinoma a challenge both clinically and pathologically. Immunohistochemical and electron microscopic examinations are useful to establish the appropriate diagnosis in such cases.     

The UTX gene escapes X inactivation in mice and humans

We recently have identified a ubiquitously transcribed mouse Y chromosome gene, Uty , which encodes a tetratricopeptide repeat (TPR) protein. A peptide derived from the UTY protein confers H-Y antigenicity on male cells. Here we report the characterization of a widely transcribed X-linked homologue of Uty , called Utx , which maps to the proximal region of the mouse X chromosome and which detects a human X-linked homologue at Xp11.2. Given that Uty is ubiquitously transcribed, we assayed for Utx expression from the inactive X chromosome (Xi) in mice and found that Utx escapes X chromosome inactivation. Only Smcx and the pseudoautosomal Sts gene on the mouse X chromosome have been reported previously to escape inactivation. The human UTX gene was also found to be expressed from Xi. We discuss the significance of these data for our understanding of dosage compensation of X-Y homologous genes in humans and mice.      

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Limiting factors for cytopathological diagnosis of high-grade squamous intraepithelial lesions: a cytohistological correlation between findings in cervical smears and loop electrical excision procedure.

The present study sought possible factors leading to the cytological diagnosis of atypical squamous cells of uncertain significance (ASCUS) in cases of high-grade squamous intraepithelial lesions (HSIL). Based on retrospective histopathological analysis of loop electrical excision procedure (LEEP) products that diagnosed HSIL, two study groups were randomly selected. The first was consisted of cases with two consecutive Papanicolaou (Pap) smears with the diagnosis of ASCUS. The second (control) group was represented by cases diagnosed as HSIL by cytology. From the Pap smears diagnosed as ASCUS, the sampling limitations was different from control group (P < 0.05). The median size of the largest lesion in each case with ASCUS was 2.66 mm (+/- 1.71 mm). In the control group, the median size of the largest lesion was 5.15 mm (+/-2.58 mm) (P < 0.05). The size of the lesion and sample limitations led patients with cervical intraepithelial neoplasms to be diagnosed as ASCUS for two consecutive times, after a 6-mo period.     

Sequence specificity of mutations induced by benzo[a]pyrene-7,8-diol-9,10-epoxide at endogenousaprt gene in CHO cells

We have determined the spectrum of mutations induced, by±trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene (BPDE) at the endogenous aprt locus in an hemizigous Chinese hamster ovary cell line exposed to 0.7 M BPDE. Southern analysis of 59 independent mutants revealed no major genomic alterations, indicating that gene inactivation was the result of a point mutation. This conclusion was confirmed by the cloning and sequencing of 21 of these mutants. The predominant mutation, the GCTTA transversion, comprised 62% of the spectrum, but other base pair substitutions and frameshifts were recovered. An examination of the target sequences for BPDE mutation revealed that mutations were localized within runs of GC base pairs. However, approximately half of these GC runs involved a particular sequence—a run of guanines flanked by adenine residues. Of seven such sites within the coding sequence ofaprt, mutations were clustered within five of them. This class of sequence occurs at codon 61 of the human C-Ha-ras1 protooncogene and may account for the selective activation of this codon by BPDE.