Role of the B domain for factor VIII and factor V expression and function

Factor V and factor VIII are homologous cofactors in the blood coagulation cascade that have the domain structure A1-A2-B-A3-C1-C2, of which the B domain has extensively diverged. In transfected COS-1 monkey cells, expression of factor VIII is approximately 10-fold less efficient than that of factor V, primarily because of inefficient protein secretion and, to a lesser extent, reduced mRNA expression. To study the functional significance and effect of the B domain on expression and activity, chimeric cDNAs were constructed in which the B domains of factor V and factor VIII were exchanged. Expression of a factor VIII chimera harboring the B-domain of factor V yielded a fully functional factor VIII molecule that was expressed twofold more efficiently than wild-type factor VIII because of increased mRNA expression. Thus, sequences within the factor VIII B domain were not responsible for the inefficient secretion of factor VIII compared with factor V. Expression of a factor V chimera harboring the B domain of factor VIII was slightly reduced compared with wild-type factor V, although the secreted molecule had significantly reduced procoagulant activity correlating with dissociated heavy and light chains and resistance to thrombin activation. Interestingly, the factor V chimera containing the factor VIII B domain was efficiently activated by Russell's viper venum (RVV). A factor V B domain deletion (residues 710- 1545) molecule also exhibited significantly reduced procoagulant activity caused by resistance to thrombin cleavage and activation, although this molecule was activatable by RVV. These results show that, in contrast to factor VIII, thrombin activation of factor V requires sequences within the B domain. In addition, thrombin activation of factor V occurs through a different mechanism than activation by RVV.     

PADGEM (GMP140) is a component of Weibel-Palade bodies of human endothelial cells

PADGEM protein (PADGEM), also known as GMP140, is a platelet alpha- granule membrane protein that is translocated to the external membrane after platelet activation. Although the biosynthesis of this protein was originally thought to be confined to megakaryocytes, the synthesis of PADGEM in endothelial cells was recently demonstrated (McEver et al: Blood 70:1974a, 1987). We now describe the subcellular localization of this protein in endothelial cells. Immunofluorescence staining of permeabilized human umbilical vein endothelial cells with KC4, a well characterized monoclonal antibody to PADGEM, showed positively stained elongated structures similar in distribution and shape to Weibel-Palade bodies. Their identity as Weibel-Palade bodies was confirmed by double label immunofluorescence using KC4 and a polyclonal antiserum to von Willebrand factor (vWf), a protein known to be specifically stored in these organelles. All Weibel-Palade bodies were found to contain PADGEM. In contrast to strong perinuclear staining produced with anti- vWf antibodies, no significant perinuclear staining was obtained with KC4, indicating that relatively little PADGEM is present in the endoplasmic reticulum and in the Golgi apparatus. In endothelial cells treated with secretagogues that stimulate vWf release the elongated structures positive for PADGEM disappeared, further identifying these structures as Weibel-Palade bodies. This observation extends the parallels between Weibel-Palade bodies and alpha-granules and suggests a possible functional association between vWf and PADGEM.     

Occurrence of the t(2;5)(p23;q35) in non-Hodgkin's lymphoma

Primary CD30(Ki-1)-positive anaplastic large-cell lymphoma (ALCL) is considered by some to be a distinct clinicopathologic entity associated with the t(2;5) (p23;q35). However, the specificity of t(2;5) for ALCL has not been carefully studied. Therefore, we performed a detailed analysis of all cases of ALCL with abnormal cytogenetics results in the Nebraska Lymphoma Study Group registry, as well as all other cases of non-Hodgkin's lymphoma with t(2;5) in the registry. We found the t(2;5) in only five of 10 cases of ALCL, four of whom were young patients. However, we also found the t(2;5) in 11 other cases of nonanaplastic lymphoma, including eight children with typical peripheral T-cell lymphomas of various types. The t(2;5) was also found in three older adults with B-cell lymphomas of various types. Thus, the t(2;5) was not specific for CD30+ ALCL. However, t(2;5) may define a clinicopathologic entity in children and young adults characterized by variable morphologies with a T-cell or indeterminate phenotype, CD30-positivity, nodal disease with frequent extranodal involvement, advanced stage, and an excellent response to therapy, including bone marrow transplantation for relapsed disease. The clinical relevance of the t(2;5) in older patients requires further study.     

Allocation of health care resources in the neonatal and perinatal area �CCPS Symposium 1996

There have been publically expressed concerns about the costs and allocation of neonatal and perinatal health care resources in Canada and elsewhere for the past 15 years. This paper reports information from a symposium held during the 1996 Canadian Paediatric Society (CPS) annual meeting sponsored by the CPS Section on Perinatal Medicine. Experts in perinatal epidemiology, health care economics, public policy and finance, and consumer perspectives on the outcomes of neonatal and perinatal intensive care explored the following questions: How should the need for health care resources in the neonatal and perinatal area be objectively determined? When there are competing needs between the maternal-newborn area and other areas, how should these be rationalized? What evidence should be used (or should be available) to support the present use of resources? What evidence should be available (or is needed) to change or introduce new uses of resources? The conclusions indicated that there are no generally accepted methods to determine the allocation of health care resources but that considerations need to include population characteristics, desired outcomes, achievable results, values, ethics, legalities, cost-benefit analyses and political objectives. Information from families and adolescents who required the use of high technology and/or high cost programs will contribute individual, family and societal values that complement cost-efficacy analyses.     

Parathyroid imaging: comparison of double-tracer (T1-201, Tc-99m) scintigraphy and high-resolution US

Parathyroid scintigraphy using a double-tracer (T1-201, Tc-99m) subtraction technique depicted 17 of 23 (74%) parathyroid adenomas in patients with and without previous neck operations. High-resolution (10-MHz) ultrasound (US) depicted 18 (78%) of these adenomas. Average tumor size depicted by US was 17 X 10 X 8 mm (excluding a giant adenoma) and 19 X 10 X 9 mm by scintigraphy. Alone, neither modality was particularly sensitive in the depiction of primary hyperplasia of the parathyroid glands, but combined techniques were more effective than the use of a single modality. With both US and T1-201 scintigraphy, only two of 23 cases of parathyroid adenoma in the neck were missed, and none of the eight cases of secondary hyperplasia were missed. In 11 patients who had previously undergone neck surgery, parathyroid tumors were identified in eight by either US or double-tracer scintigraphy. Preoperative parathyroid imaging with double-tracer scintigraphy and high-resolution US is suggested for patients with hyperparathyroidism, particularly in those patients who have had previous parathyroid surgery.     

Intrauterine growth retardation: evaluation by magnetic resonance. Work in progress

Eleven high risk fetuses between 32 and 37 menstrual weeks gestational age were examined by magnetic resonance (MR) imaging. Serial obstetrical sonograms, birth weights, and serial postnatal examinations were obtained in all subjects. Sagittal MR spin echo images obtained using TR = 0.5 sec and TE = 28 msec were useful for assessing subcutaneous fat. Prospective estimates of fetal fat stores correlated with neonatal outcome better than sonographic measurements of fetal growth parameters or actual birth weight. MR appears to be a safe and useful technique that offers information complementary to obstetrical sonography when IUGR is suspected.     

Phage typing in dermatitis cruris pustulosa et atrophicans: does staphylococcal carrier status have a role?

Background Dermatitis cruris pustulosa et atrophicans (DCPA) is a form of chronic folliculitis of the legs with a multifactorial etiopathogenesis, seen primarily in tropical countries. Staphylococcus aureus has been isolated from the pustules in earlier studies, although the organisms isolated have not been further characterized. Materials and methods Patients with DCPA, who attended the Dermatology outpatient clinic at JIPMER, Pondicherry, India, during the study period (December 2006–June 2008) were included. Pus from the lesions as well as swabs from carrier sites (nares, axillae, and gluteal fold) were cultured. Staphylococcus aureus isolates were subjected to phage typing at the National Staphylococcal Phage Typing Center, Department of Microbiology, Maulana Azad Medical College, New Delhi, India. Results Thirty‐seven patients were included in the study. Pus from the folliculitic lesions grew S. aureus in 32 (86.49%) patients. Based on the comparison of antibiotic sensitivity patterns, isolates from pus and carrier sites were found to be similar in 15 patients. Phage typing established the organism to be identical in five of these patients. Conclusions Characterization of S. aureus in DCPA shows that there is no specific phage type that is uniformly responsible for the lesions in most patients. However, in view of the unclear etiology of this condition, the pathogenicity of a staphylococcal carrier state in individual patients needs to be addressed.     

Squamous cell carcinoma complicating epidermolysis bullosa in a 6-year-old girl

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare form of epidermolysis bullosa (EB) that presents with generalized blistering since birth. Squamous cell carcinoma (SCC) is the most common cutaneous malignancy seen in RDEB, starting from second decade onwards. We report a case of SCC complicating RDEB in a 6-year-old girl.     

Surgical sperm retrieval and intracytoplasmic sperm injection as treatment of obstructive azoospermia

Male genital tract obstructions may result from infections, previous inguinal and scrotal surgery (vasectomy) and congenital bilateral absence of the vas deferens (CBAVD). Microsurgery can sometimes be successful in treating the obstruction. In other cases and in cases of failed surgical intervention, the patient can be treated by microsurgical or percutaneous epididymal sperm aspiration (MESA, PESA) or testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI). We present the results of 39 ICSI procedures for obstructive azoospermia in 24 couples. The aetiology of the obstruction was failed microsurgery in 11 patients, CBAVD in nine and genital infections in four. Sperm retrieval was accomplished via MESA in four cases, PESA in 18 cases and via TESE in 11 cases. TESE was only applied when PESA failed to produce enough spermatozoa for simultaneous ICSI. In six patients, the ICSI procedure was performed with cryopreserved spermatozoa after an initial PESA procedure. Fertilization occurred in 47% of the metaphase II oocytes; embryo transfer was performed in 92% of procedures and resulted in a clinical pregnancy in 13/39 procedures. Ongoing pregnancy was achieved in 10/39 procedures. One pregnancy was terminated early after prenatal investigation showed a cytogenetic abnormality (47,XX+18, Edwards syndrome). The other nine pregnancies resulted in the live birth of 10 children, without any congenital abnormalities. Epididymal and testicular retrieved spermatozoa were successfully used for ICSI to treat obstructive azoospermia, and resulted in an ongoing pregnancy in 10 of 24 couples (41.6%) after 39 ICSI procedures, a success rate of 25.6% per treatment cycle and of 27.7% per embryo transfer.      

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.