The tyrosine-based YXXØ targeting motif of murine leukemia virus envelope glycoprotein affects pathogenesis

Retroviruses, such as human and simian immunodeficiency viruses (HIV and SIV), and murine leukemia viruses (MuLV), harbor a tyrosine-based motif in the intracytoplasmic domain of their envelope glycoprotein. This motif can act as an endocytosis signal or as a targeting signal, restricting viral budding at specific cell surface membrane domains. In the present study, proviral DNA of the ecotropic Cas-Br-E strain of MuLV was modified by substitution or deletion of the critical tyrosine residue. Mutant viruses lost basolateral targeting in polarized MDCK epithelial cells while expression level of the glycoprotein at the cell surface was not affected. This suggests that the tyrosine-based motif in MuLV does not act as an endocytosis signal. Only a small delay in the appearance of disease was observed in inoculated mice. In contrast, a striking change in the pathology was observed with enlarged thymus and lymph nodes in animals inoculated with mutant viruses.     

Clinical and molecular study in congenital muscular dystrophy with partial laminin alpha 2 (LAMA2) deficiency

Complete laminin alpha2 (LAMA2) deficiency causes approximately half of congenital muscular dystrophy (CMD) cases. Many loss-of-function mutations have been reported in these severe, neonatal-onset patients, but only single missense mutations have been found in milder CMD with partial laminin alpha2 deficiency. Here, we studied nine patients diagnosed with CMD who showed abnormal white-matter signal at brain MRI and partial deficiency of laminin alpha2 on immunofluorescence of muscle biopsy. We screened the entire 9.5 kb laminin alpha2 mRNA from patient muscle biopsy by direct capillary automated sequencing, single strand conformational polymorphism (SSCP), or denaturing high performance liquid chromatography (DHPLC) of overlapping RT-PCR products followed by direct sequencing of heteroduplexes. We identified laminin alpha2 sequence changes in six of nine CMD patients. Each of the gene changes identified, except one, was novel, including three missense changes and two splice-site mutations. The finding of partial laminin alpha2 deficiency by immunostaining is not specific for laminin alpha2 gene mutation carriers, with only two patients (22%) showing clear causative mutations, and an additional three patients (33%) showing possible mutations. The clinical presentation and disease progression was homogeneous in the laminin alpha2-mutation positive and negative CMD patients.     

Physicochemical model of alginate-poly-L-lysine microcapsules defined at the micrometric/nanometric scale using ATR-FTIR, XPS, and ToF-SIMS

Alginate-poly-L-lysine-alginate (APA) microcapsules are currently being investigated as a means to immuno-isolate transplanted cells, but their biocompatibility is limited. In this study, we verified the hypothesis that poly-L-lysine (PLL), which is immunogenic when unbound, is exposed at the APA microcapsule surface. To do so, we analysed the microcapsule membrane at the micrometric/nanometric scale using attenuated total reflectance Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and time-of-flight secondary ion mass spectrometry. The results indicate that PLL and alginate molecules interact within the membrane. PLL exists in considerable amounts near the surface, contributing to the majority of the carbon within the outermost 100 Angstroms of the membrane. PLL was also detected at the true surface (the outermost monolayer) of the microcapsules. The exposure of PLL does not appear to result from defects in the outer alginate coating. This physicochemical model of APA microcapsules could explain their immunogenicity and will play an important role in the optimization of the microcapsule design.     

Diminished neo-antigen response to keyhole limpet hemocyanin (KLH) vaccines in patients after treatment with chemotherapy or hematopoietic cell transplantation

Relapse is the most common cause of treatment failure for advanced cancer, even those treated with autologous hematopoietic cell transplantation (HCT). Effective tumor-specific immunotherapy may decrease relapse, however, this will fail if the immune system is unable to respond. We developed a strategy to test immune responses with a single injection of the bona fide neo-antigen KLH. The model was first tested in 37 normal volunteers using three KLH vaccines: Intracel KLH, Biosyn KLH, and Biosyn KLH + adjuvant. Despite finding the immunogenic epitope conserved in both products, intact Intracel KLH induced a better response compared to a purified 350/390 kDA subunit of KLH contained in the Biosyn KLH product. Addition of a synthetic oil adjuvant (Montanide ISA51) restored the response to a single injection of Biosyn KLH. A quantitative readout measured by a KLH-specific cellular and humoral response with isotype switching 1 month after KLH vaccination was established. To test the integrity of the adaptive immune response in cancer patients, we vaccinated 14 patients post-HCT and 19 patients with advanced cancer with KLH vaccines that elicited a 100% response rate in normal volunteers. In marked contrast to normal subjects, both responses were significantly impaired up to 16 months after autologous HCT with an intermediate response in advanced cancer patients. KLH vaccines are safe and require only a single injection to test neo-antigen responses providing an optimal platform for definitive testing of strategies to improve diminished immune recovery after chemotherapy or post-HCT.     

Detection of infectious human immunodeficiency virus type 1 in female genital secretions by a short-term culture method

Infectious human immunodeficiency virus type 1 (HIV-1) is difficult to detect in female genital secretions by standard virus culture techniques. To improve detection of cell-free HIV-1 in female genital secretions, we adapted a short-term assay that uses the multinuclear-activation galactosidase indicator (MAGI) assay. When vaginal lavages from HIV-1-infected women were tested with the adapted MAGI assay, 25 (64%) of 39 lavages with detectable, cell-free HIV-1 RNA were shown to have infectious virus. No infectious virus was found in 10 vaginal lavages from HIV-1-infected women with undetectable vaginal viral loads. Significantly (P < 0.01) more lavages from HIV-1-infected women tested positive for infectious virus by the MAGI assay than by standard peripheral blood mononuclear cell (PBMC) coculture, which detected infectious virus in only 6 (17%) of 35 vaginal lavages. Lavages with viral loads of >10,000 copies per lavage yielded significantly (P < 0.01) more positive cultures than those with <10,000 copies by using the MAGI assay. Detection of infectious HIV-1 in vaginal lavages was not associated with the presence of genital tract infections or CD4(+)-T-cell counts. However, although the results were not significant (P = 0.08), the MAGI assay detected infectious virus from more vaginal lavages at a vaginal pH of >/=4.5 than at a pH of <4.5. These results indicate that the MAGI assay is more sensitive than PBMC culture methods for detecting infectious virus in female genital secretions. Accurate measurements of infectious virus in genital secretions will improve studies that evaluate sexual transmission of HIV-1.     

Gluthatione-S-transferase P1 polymorphism I105V in familial and sporadic prostate cancer

Several reports suggest that the glutathione-S-transferase (GST) family of enzymes is involved in a variety of cancers, due to their carcinogen-detoxification properties. A polymorphism in codon 105 of the pi variant (GSTP1 I105V), which affects the enzymatic activity of the enzyme, has been linked to the incidence of cancers from different organs. However, the published data in prostate cancer (PCa) is controversial. Some studies report an association with the GSTP1 I105V polymorphism and sporadic PCa, whereas other studies report no association. Recently, one study showed a positive correlation between the GSTP1 I105V polymorphism and familial PCa in a Japanese population. In the present study, we assessed the correlation of the GSTP1 I105V polymorphism with familial and sporadic PCa in an American population. We analyzed DNA samples from 438 patients with familial PCa, 499 patients with sporadic PCa, and 510 controls. We found no significant association between the GSTP1 I105V polymorphism and familial or sporadic PCa when compared to the control group [odds ratio (OR) =1.0 (0.74-1.37); P=0.58]. Moreover, no association was found after stratification for age of diagnosis, Gleason grade, or lymph node involvement [OR =0.84 (0.65-1.09), P=0.37]. These data indicate that there is no associated risk for sporadic or familial PCa in American families containing the GSTP1 I105V polymorphism.     

Complete overlap of PHACE syndrome and sternal malformation--vascular dysplasia association

PHACE syndrome is the term applied to the association of posterior fossa brain abnormalities, hemangiomas, arterial anomalies in the cranial vasculature, coarctation of the aorta/cardiac defects, and eye abnormalities. An overlap with the sternal malformation/vascular dysplasia association has been described. We report an adult patient with complete manifestations of both conditions. As an adult she has demonstrated resolution of the hemangiomas and only mild intellectual difficulties.