Site-directed ELISA with synthetic peptides representing the HIV transmembrane glycoprotein

Two partially overlapping 19 and 22 amino acids long peptides representing a highly immunogenic site of the transmembranous glycoprotein (gp41) of human immunodeficiency virus (HIV) were used as antigen in ELISA tests. The results of antibody determination with this assay were compared with those of three or more conventional ELISAs and Western blot (WB) tests and radioimmunoprecipitation assay. Twenty-six sera from patients with AIDS or LAS and from asymptomatic carriers of HIV infection all showed a pronounced reaction in the peptide ELISA as well as positive results with other tests. In contrast, 27 sera from laboratory workers and blood donors were negative by all tests. A group of 39 blood donor sera, which had shown false positive or ambiguous results in the ELISAs and sometimes in WB tests employed for confirmation, also were negative in all cases with the peptide ELISA. Consecutive samples collected from individuals with primary HIV infection were also analyzed. In 6 out of 9 cases, the peptide ELISA revealed an antibody response within one month after onset of clinical symptoms and sensitivity for antibody detection equaled that of other ELISA tests. Eight sera from five West African persons infected with HIV-related viruses did not react in the peptide ELISA, reflecting differences in properties of the envelope components. The peptide ELISA used in this study appears to represent a simple technique employing chemically synthesized antigen for accurate and sensitive estimation of antibodies to the HIV group of nontransforming human retroviruses.     

Apoptosis induction and interferon signaling but not IFN-beta promoter induction by an SV5 P/V mutant are rescued by coinfection with wild-type SV5

Infection of human cells with the paramyxovirus simian virus 5 (SV5) results in minimal cytopathic effect, and host interferon (IFN) and apoptotic pathways are not activated. We have previously shown that an rSV5 containing six naturally occurring P/V gene substitutions (rSV5-P/V-CPI-) displays premature and elevated expression of viral RNA and protein. In addition, cells infected with rSV5-P/V-CPI- show induction of the IFN-beta promoter as well as activation of IFN signaling and apoptotic pathways. In this article, we have tested the hypothesis that rSV5-WT can supply trans-acting factors that prevent host cell antiviral responses induced by rSV5-P/V-CPI-. During coinfection of human A549 cells, rSV5-WT blocked cell rounding, loss of cell volume, and DNA fragmentation induced by rSV5-P/V-CPI-, three later events in the apoptotic pathway, but was not able to block the loss of mitochondrial membrane potential (DeltaPsi(m)), an early event in the cell death process. As expected, IFN signaling was blocked during coinfections, and this was attributed to the loss of STAT1 induced by the rSV5-WT V protein. Surprisingly, simultaneous infection with rSV5-WT could not suppress the activation of the IFN-beta promoter by rSV5-P/V-CPI- infection. However, the IFN-beta promoter was not activated in cells that were first preinfected for 1 h with rSV5-WT and then subsequently infected with rSV5-P/V-CPI-. A model is proposed for activation of host responses to infection with the rSV5-P/V-CPI- mutant and the steps that are blocked by rSV5-WT.     

Defects in neurofibromatosis 2 protein function can arise at multiple levels

Neurofibromatosis 2 (NF2) is an inherited cancer syndrome resulting from mutations in the NF2 tumor suppressor gene. Analysis of NF2 mutations has revealed some general genotype-phenotype correlations. Severe disease has been associated with mutations that produce a premature termination while more mild disease has been associated with missense mutations. Here, we provide experimental proof for these genotype-phenotype correlations by demonstrating that nonsense mutations fail to produce stable merlin protein while missense mutations result in the generation of merlin proteins defective in negative growth regulation. This inability to suppress cell growth may result from defects in the function of merlin at several levels, including failure to form an intramolecular complex. Based on these findings, we propose a model for merlin growth suppression that provides a framework for analyzing NF2 patient mutations and merlin function.      

Growth sensitivity of a recombinant simian virus 5 P/V mutant to type I interferon differs between tumor cell lines and normal primary cells

A paramyxovirus SV5 mutant (rSV5-P/V-CPI-) that encodes 6 naturally-occurring P/V gene substitutions is a potent inducer of type I interferon (IFN) and is restricted for low moi growth, two phenotypes not seen with WT SV5. In this study, we have compared the IFN sensitivity of WT SV5 and the rSV5-P/V-CPI- mutant in tumor cell lines and in cultures of normal primary cells. We have tested the hypothesis that differences in IFN induction elicited by WT rSV5 and rSV5-P/V-CPI- are responsible for differences in low moi growth and spread. In contrast to WT SV5, low moi infection of A549 lung carcinoma cells with rSV5-P/V-CPI- resulted in a plateau of virus production by 24-48 h pi when secreted IFN levels were between approximately 100 and 1000 U/ml. Gene microarray and RT-PCR analyses identified IFN genes and IFN-stimulated genes whose expression were increased by infection of A549 cells with WT and P/V mutant viruses. Restricted low moi growth and spread of rSV5-P/V-CPI- in A549 cells was relieved in the presence of neutralizing antibodies to IFN-beta but not TNF-alpha. When A549 or MDA-MB-435 breast tumor cells were pretreated with IFN, both WT and P/V mutant viruses showed delayed spread and approximately 10-fold reduction in virus yield, but infections were not eliminated. Using normal primary human epithelial cells that have undergone limited passage in culture, WT rSV5 and rSV5-P/V-CPI- displayed high moi growth properties that were similar to that seen in A549 cells. However, IFN pretreatment of these primary cells as well as normal human lung cells eliminated low moi spread of both mutant and WT rSV5 infections. Together, these data demonstrate that SV5 growth in normal primary human cells is highly sensitive to IFN compared to growth in some tumor cell lines, regardless of whether the P/V gene is WT or mutant. These results suggest a model in which spread of WT SV5 in normal human cells is dependent on the ability of the virus to prevent IFN synthesis. The implications of these results for the use of recombinant paramyxoviruses as vectors are discussed.     

Comparison of the structure of human intervertebral discs in the cervical,thoracic and lumbar regions of the spine

Posterior and anterior heights, cross-sectional area and shape were measured for all the intervertebral discs in four spines from elderly human cadavers. Disc height was a minimum at the T4-5 level; thoracic discs were less wedge-shaped than those in the cervical and lumbar regions. Cross-sectional area increased from the cranial to caudal extremity; at the L5-S1 level the nucleus pulposus occupied a high proportion of this area. Cervical discs tended to have an elliptical cross-sectional shape, thoracic discs were more circular and lumbar discs tended to have an elliptical cross-section which was flattened or re-entrant posteriorly. This shape distribution was quantified by defining a shape index which had a maximum value of 1 for a circular cross-section. Orientations of the reinforcing fibres in the outer lamellae of the anterior annulus fibrosus were measured from 27 discs by X-ray diffraction. For these measurements, C3-4, T7-8 and L2-3 were chosen as representative of cervical, thoracic and lumbar discs. The fibre tilt, with respect to the axis of the spine, was significantly less in the cervical discs (at 65 degrees) than in the thoracic and lumbar discs (about 70 degrees). These findings are interpreted in relation to differing functional requirements and possible mechanisms of failure in the cervical, thoracic and lumbar regions of the spine in the light of current knowledge on the biomechanics of the intervertebral disc.     

Separating fact from fiction: assessing the potential of modified adenovirus vectors for use in human gene therapy

One of the major hurdles to successful gene therapy of genetic and/or acquired disease is the ability to efficiently introduce a foreign gene into the tissue of interest and, in the case of some genetic diseases, achieve long-term expression of the transgene. Due to their ability to transduce a wide variety of cell types in a cell-cycle independent fashion, adenovirus (Ad)-based vectors have received considerable attention in recent years as delivery vehicles for multiple gene therapy applications. Effective use of early "first-generation" versions of these vectors was hampered by not only the induction of strong immune responses in the host to the Ad vector and transduced cells, but also to direct acute and chronic toxicity caused by the vector itself. Furthermore, transgene expression was typically transient, lasting only a few weeks. Despite these limitations, these vectors have been used in a number of human clinical trials, eliciting both interesting as well as controversial results, some of which are summarized herein. Because of these limitations, a number of advances in adenovirus "vectorology", manifested primarily as the development of multiply attenuated Ads and vectors deleted of all viral protein coding sequences, has resulted in vectors which retain all of the advantages of Ad vectors and, in addition, do not exhibit the deleterious characteristics associated with [E1-]deleted Ads. This review focuses on the current state of the art regarding the potential for human use of Ad-based vectors, and how the use of this vector continues to offer the potential for successful use as a gene delivery tool for the treatment of a great number of human genetic and non-genetic diseases.     

Detecting pre-ovulatory luteinizing hormone surges in urine

The study objectives were to determine (i) if pre-ovulatory luteinizing hormone (LH) surges, undetected in urine by two immunoradiometric assays (IRMA), were detectable by an ultrasensitive immunofluorometric assay (IFMA) and (ii) the influence of creatinine adjustment on the detection and timing of the urinary LH surges. Daily urine specimens were contributed by healthy 25-36 year old volunteers during 14 ovulatory menstrual cycles for an epidemiological study conducted in 1983-1985. Specimens were selected as having been previously assayed by two IRMA without consistently detecting LH surges. These urine specimens were remeasured using an IFMA and adjusted for creatinine concentration. IFMA measurements revealed unambiguous LH surges in all cycles. Adjusting IRMA urinary LH values for creatinine concentrations revealed previously undetected LH surges in four of eight cycles. Creatinine adjustment also altered the timing of IRMA and IFMA LH surges by 1-5 days. These results demonstrate an IFMA that detects pre- ovulatory LH surges in unpreserved, frozen urine from cycles where such surges were previously undetectable. Further, creatinine adjustment can markedly affect detection and timing of the onset and peak of the urinary LH surge. While our analysis suggests that this adjustment improves the validity of the LH measure, this requires further investigation.