Formaldehyde in cryoprotectant propanediol and effect on mouse zygotes

Cryopreservation of human zygotes and embryos has been routinely performed by in-vitro fertilization clinics for many years. Karran and Legge (1996) first reported that formaldehyde (FA) present in the cryoprotective solutions can have a deleterious effect on mouse oocytes. FA is a cytotoxic, carcinogenic and mutagenic chemical. The effect of FA on mouse zygotes was investigated. In addition, the concentrations of FA in propanediol (PROH) obtained from various sources were determined. Pooled 1-cell embryos were dispensed into droplets of modified Ham's F10 or human tubal fluid containing various concentrations of FA. Since bovine serum albumin (BSA) may minimize toxicity additional trials were done as above in the absence of BSA. FA concentration in the standard 1.5 M PROH, from different sources in water, was measured in the same assay using a standard curve of 0-100 microM FA. FA in a complex medium had a significant deleterious effect on embryo development and hatching but only at 1 mM concentration (P < 0.000001; see Tables I-III). There was no significant effect of FA at 100 microM. However, in a simple medium even 50 microM FA decreased embryo hatching. FA was present in 1.5 M PROH from different sources (range 1.0-35.3 microM concentration). It appears that FA concentrations do not increase with storage because FA concentrations were low even after opening and storage for 3 years on the shelf. This suggests that FA is a contaminant during the manufacturing process and may vary from manufacturer to manufacturer and batch to batch. Until further studies are done to confirm the lack of toxicity to embryos during cryopreservation (with or without FA scavengers) it may be prudent to screen all batches of cryoprotectants for FA as part of quality control.      

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.      

Oocyte morphology predicts outcome of intracytoplasmic sperm injection

To examine the influence of cytoplasmic morphology on the success rate of intracytoplasmic sperm injection (ICSI), the morphology of 837 metaphase II oocytes was assessed after cumulus stripping. The main abnormalities detected were excessive granularity, cytoplasmic inclusions such as vacuoles, smooth endoplasmic reticulum clustering and refractile bodies. Microinjection was performed in 538 oocytes with normal cytoplasm, 142 out of 161 with excessive granularity and 112 out of 138 with cytoplasmic inclusions. Very poor oocytes were not injected. No difference was found in fertilization rate. The embryos achieved cleaved normally and a similar number of good quality embryos among the three groups was noted. The outcome of transfer of embryos derived solely from normal oocytes (group A: 72 patients, 183 embryos) was compared with those from oocytes with cytoplasmic abnormalities (group B: 34 patients, 85 embryos). In group A, 17 clinical pregnancies (24% per patient, implantation rate 10%) were established. In group B, only one clinical pregnancy (3% per patient, implantation rate 1%) was established, from the transfer of embryos derived from oocytes with homogeneous granularity of the cytoplasm. No pregnancy resulted following the transfer of embryos from eggs with cytoplasmic inclusions. The difference was statistically significant. The outcome of ICSI is dependent on the quality of the oocytes retrieved. Normal fertilization and early embryo development were achieved in oocytes with abnormal cytoplasm morphology, but the resulting embryos failed to demonstrate the same implantation potential as those derived from oocytes with normal cytoplasm.      

<Emphasis Type="Italic">Trypanosoma evansi</Emphasis> in inbred and Swiss-Webster mice: distinct aspects of pathogenesis

Trypanosoma evansi (Trypanosomatidae, Kinetoplastida) is a salivarian trypanosomatid that infects eight mammal orders spread over America, Europe and Asia. In Brazil, T. evansi is the etiological agent of Mal de Cadeiras, a horse disease very often described in the region known as Pantanal do Mato Grosso. Few data concerning the genetic diversity and biology of subpopulations of T. evansi that circulate in Brazil are available. The factors that modulate the interaction of this parasite with its hosts also remain to be elucidated. Here we evaluated the course of experimental infection of six T. evansi isolates derived from domestic and wild animals in Swiss-Webster mice and three Mus musculus lineages. The follow-up included biological, immunological as well as biochemical and hematological parameters. The same isolates as well as three others were characterized by pulsed-field electrophoresis. Our results showed that T. evansi isolates displayed significant differences regarding behavior and morbidity patterns in the distinct mouse lineages. Nevertheless, these differences could not be correlated with pulsed-field electrophoresis profiles. Indeed, concerning this molecular marker, only microheterogeneity was observed. Moreover, we observed that the outcome of the infection is defined by both host genetic background and peculiarities (virulence factors) of the distinct T. evansi isolates. Anemia and hypoglycemia were the only features that could be observed in all mouse lineages, independently of the inoculated T. evansi subpopulation. In addition, our data also show that Mus musculus is a suitable model host for the study of the different pathogenetic features of T. evansi infection.     

Intermediate filament expression by normal and diseased human corneal epithelium

Cicatricial conjunctivitis may be a sequel to systemic disorders (eg, Stevens-Johnson syndrome, cicatricial pemphigoid) or local disorders such as chemical burns. The cicatrisation is often associated with corneal epithelial changes that cause visual loss. These have been attributed to encroachment of the conjunctival epithelium over the cornea. However, the epithelial anomalies are poorly understood. We investigated the corneal epithelial changes in cicatricial conjunctivitis with an immunohistochemical study of intermediate filaments in normal and pathological specimens. Our results show that the normal corneal epithelium is immunoreactive for cytokeratin 3 (CK 3) but not cytokeratin 19 (CK 19), whereas normal conjunctival epithelium is CK 3 negative and CK 19 positive. Conjunctiva artificially transposed over the cornea (after therapeutic conjunctival flap reconstruction) retained the normal pattern of conjunctival cytokeratin expression (CK 3 negative, CK 19 positive). Conversely, the entire corneal epithelium exhibited the normal cytokeratin pattern (CK 3 positive, CK 19 negative) in 82% of Stevens-Johnson, 80% of cicatricial pemphigoid, and 69% of chemical burns specimens. The findings suggest that conjunctival encroachment is not responsible for the changes at the corneal surface in cicatricial conjunctivitis and that the abnormal corneal epithelium is derived from native corneal cells in these diseases.     

Donor Choriocarcinoma Transmission From Solid Organ Transplantation: A Case Report

Transplantation of any organ has some inherent risk of disease transmission, such as infection and malignancy. The present study aims to describe 2 cases of choriocarcinoma transmission after kidney and liver transplantation originating from the same patient. The donor was a 17-year-old woman who died of cerebral hemorrhage. Both organ recipients died of metastatic choriocarcinoma few months after the transplantation, within days after starting chemotherapy. Retrospective hCG (human chorionic gonadotropin hormone) analysis in donor's blood stored at the time of donation had a result of 9324 mIU/mL. Despite its rarity, clinicians should be aware of the risk of transplant-related choriocarcinoma from female donors in childbearing age. In some cases, hCG dosage should be performed before donation.     

乳头腺管口挑拨治疗急性乳腺炎32例

0　引言　急性乳腺炎是产后妇女的常见病 .我们采用针头挑拨治疗急性乳腺炎 32例 ,疗效显著 .1　临床资料　患者 32例 ,年龄 2 3～ 34 (平均 2 5 .3)岁 ,均为产后哺乳产妇 .其中患乳胀痛为共有症状 .伴发热 10例 ;右侧乳腺炎 14例 ,左侧乳腺炎 16例 ,双侧乳腺炎 2例 ;外上象限胀痛 6例 ,外下象限胀痛 8例 ,内上象限胀痛 7例 ,内下象限胀痛 6例 ,二象限同时胀痛 5例 .乳头均无破损 .患者取卧位 ,显露患乳 ,乳头以碘伏严密消毒后 ,术者持五号针头以指腹常规触诊患乳 ,触摸到肿胀部位后 ,沿乳腺管走行方向找到对应的乳头腺管口 ,以针尖轻轻挑拨…