OBJECTIVE: To determine the sensitivity, specificity, and negative and positive predictive values of cerebrospinal fluid (CSF) xanthochromia for detection of ruptured cerebral aneurysms in patients with thunderclap headache (sudden and severe headache with maximal intensity at onset). PATIENTS AND METHODS: We identified patients who presented to our emergency department with symptoms of thunderclap headache between January 1, 1998, and January 1, 2008. Those with normal results on computed tomography (CT) of the head were selected for inclusion in the study. All consenting adult patients had undergone a lumbar puncture procedure for CSF analysis. Xanthochromia was assessed by visual inspection. All patients with a normal CT result were further evaluated by conventional 4-vessel catheter angiography. If no aneurysms were detected in the initial study, the procedure was performed again within 7 to 14 days. All patients were followed up clinically for a mean period of approximately 29 months. RESULTS: A total of 152 patients were identified (mean +/- SD age, 44.7+/-15.8 years; 53 men). Cerebrospinal fluid xanthochromia was present in 18 patients (12%), 13 (72%) of whom were ultimately diagnosed as having aneurysmal subarachnoid hemorrhage. A single patient who tested negative for xanthochromia and had a normal CT result was subsequently found to have a ruptured middle cerebral artery aneurysm. The sensitivity, specificity, positive predictive value, and negative predictive value of CSF xanthochromia for detection of cerebral aneurysms were 93%, 95%, 72%, and 99%, respectively. CONCLUSION: Although not perfect, visual inspection of CSF in patients with a history suggestive of subarachnoid hemorrhage remains a highly valuable and simple test to detect a recently ruptured aneurysm.

Interferons (IFNs) are potent, pleiotropic cytokines, and therefore it is likely that the cell has mechanisms to modulate IFN activity in response to excessive or prolonged IFN exposure. To investigate this question, Jurkat T cells were exposed to IFN-beta1a in vitro. The effect of dose and frequency of IFN treatment on receptor expression, the signal transduction pathway, and biologic activity was examined. Results demonstrate that at even modest doses of IFN (60 IU/ml), cell surface expression of the IFN receptor subunit, IFNAR-1, decreased significantly, and the cells were unresponsive to further IFN treatment. More interestingly, after an initial treatment with very low concentrations of IFN (<10>IU/ml), even when receptor levels remained normal and phosphorylation of signaling molecules occurred, cells were still refractory to further IFN treatment. After withdrawal of IFN, full cellular responsiveness was a progressive but surprisingly slow process. Cells retreated 2 days or 4 days after the initial IFN treatment were still refractory to even high doses (500 IU/ml) of IFN. Cells retreated 1 week after the initial IFN treatment were fully responsive. High levels of Stat1 and Stat2 correlated with the block in transcriptional activation of IFN-dependent genes and may be a mechanism by which cells can downmodulate an IFN response. Similar results were obtained when fresh peripheral blood mononuclear cells (PBMC) were treated with IFN and expression of the endogenous IFN-dependent gene, MxA, was examined. Cell surface levels of IFNAR-1 decreased and Stat1 levels increased after IFN-beta treatment, and retreatment with IFN resulted in an attenuated induction of Mx protein expression. In the context of using IFNs as therapeutic agents in the treatment of human disease, our data suggest that increasing the amount or frequency of IFN administration may not yield desired biologic effects. Thus, issues concerning the dosage and the frequency of IFN-beta administration deserve careful consideration.

The human immunodeficiency virus type-1 matrix protein (HIV-1 MA) is a multifunctional structural protein synthesized as part of the Pr55 gag polyprotein. We have used in vitro genetic selection to identify an RNA consensus sequence that specifically interacts with MA (Kd = 5 x 10(-7) M). This 13-nt MA binding consensus sequence bears a high degree of homology (77%) to a region (nt 1433-1446) within the POL open reading frame of the HIV-1 genome (consensus sequence from 38 HIV-1 strains). Chemical interference experiments identified the nucleotides within the MA binding consensus sequence involved in direct contact with MA. We further demonstrate that this RNA-protein interaction is mediated through a stretch of basic amino acids within MA. Mutations that disrupt the interaction between MA and its RNA binding site within the HIV-1 genome resulted in a measurable decrease in viral replication.

The human immunodeficiency virus type-1 matrix (HIV-1 MA) is best described as a multi-functional, structural protein. However, the multitude of functional activities ascribed to this viral component is not nearly as interesting as are its seemingly paradoxical and opposing roles during the viral life cycle. At the time of virus infection, HIV-1 MA remains associated with the reverse transcription complex, in which viral nucleic acids are synthesized, and facilitates its translocation to the host cell nucleus (Bukrinsky, Sharova et al. 1992; Bukrinsky, Sharova et al. 1993). This activity of MA has been proposed to form the basis for the infection of non-dividing cells (Bukrinsky, Haggerty et al. 1993). An interaction between the C-terminally phosphorylated form of MA and HIV-1 integrase, an integral component of the complex, was initially proposed to mediate this association (Gallay, Swingler et al. 1995; Gallay, Swingler et al. 1995). However, conditions which promote dissociation of integrase from the reverse transcription complex do not reduce MA association (Miller, Farnet et al. 1997). The possibility of a direct interaction between MA and the viral genome is discussed in Chapter III. The nucleophilic nature of HIV-1 MA is paradoxical with its reported activity in targeting the viral precursor proteins to the cytoplasmic membrane (Krausslich and Welker 1996), during the particle production phase of the viral life cycle. Furthermore, MA when expressed in the absence of other viral proteins exhibits a cytoplasmic localization (Fouchier, Meyer et al. 1997); a result which does not support a nuclear translocation role for this protein. The work presented here resolves this seemingly controversial issue. We demonstrate that MA exhibits a strong nuclear export activity. This newly discovered activity is designed to effectively counteract the protein's innate nucleophilic nature, thus maintaining a cytoplasmic localization. The nuclear export function of MA is sensitive to changes within the conformation of the protein as C- and N-terminal deletions, as well as point mutations in the protein, abolish the activity. Furthermore, the export activity is mediated by the Crm1 NES receptor (Fornerod, Ohno et al. 1997; Fukuda, Asano et al. 1997; Ossareh-Nazari, Bachelerie et al. 1997) despite the lack of a leucine-rich export signal within the matrix coding region. Therefore, the interaction between matrix protein and Crm1 is most likely to be mediated by another, perhaps cellular, protein. Any changes in matrix structure may lead to the disruption of this protein-protein interaction. We discuss a model implicating a phosphorylation event in the inactivation of this nuclear export signal. An even more fascinating issue regards the role of this nuclear export activity, during the viral life cycle, and is detailed in Chapter II. In short, mutations in MA which impair its nuclear export activity result in nuclear accumulation of the precursor Gag polyprotein (Pr55) and the nucleocapsid-associated viral genomic RNA. As a result, non-infectious virions deficient in genomic viral RNA are produced. Therefore, drugs designed to block this export activity can undermine the carefully orchestrated course of events during HIV replication and can shut down the growth of the virus.

An important aspect of the pathophysiology of human immunodeficiency virus type-1 (HIV-1) infection is the ability of the virus to replicate in non-dividing cells. HIV-1 matrix (MA), the amino-terminal domain of the Pr55 gag polyprotein (Pr55), bears a nuclear localization signal that promotes localization of the viral preintegration complex to the nucleus of non-dividing cells following virus entry. However, late during infection, MA, as part of Pr55, directs unspliced viral RNA to the plasma membrane, the site of virus assembly. How MA can mediate these two opposing targeting functions is not understood. Here we demonstrate that MA has a previously undescribed nuclear export activity. Although MA lacks the canonical leucine-rich nuclear export signal, nuclear export is mediated through the conserved Crm1p pathway and functions in both mammalian cells and yeast. A mutation that disrupts the MA nuclear export signal (MA-M4) mislocalizes Pr55 and genomic viral RNA to the nucleus, thereby severely impairing viral replication. Furthermore, we show that MA-M4 can act in a dominant-negative fashion to mislocalize genomic viral RNA even in the presence of wild-type MA. We conclude that the MA nuclear export signal is required to counteract the MA nuclear localization signal, thus ensuring the cytoplasmic availability of the components required for virion assembly.