Neurovirulence of Cryptococcus neoformans determined by time course of capsule accumulation and total volume of capsule in the brain.

Cryptococcal meningitis remains a significant opportunistic infection in HIV-infected individuals worldwide, despite availability of antiretroviral therapies in developed nations. Current therapy with amphotericin B is difficult to administer and only partially effective. Mechanisms of cryptococcal neuropathogenesis are still not clearly defined. In the present study, we used a C57Bl/6 mouse model with intravenous inoculation of three isogenic strains of Cryptococcus neoformans: H99, Cap59, and Pkr1-33. These strains differ in their capsule production and are normocapsular, hypocapsular, and hypercapsular, respectively. We studied the role of capsule in the morbidity and mortality of our host animal. Surprisingly, we found that the hypercapsular strain was least virulent while the strains that produced less capsule were more virulent and had higher concentrations of organism in the brain. These results suggest that neurovirulence is related to total capsule volume and rate of capsule accumulation in the brain, rather than the amount of capsule produced per organism. Therapies which decrease central nervous system dissemination and inhibit replication rates in the brain may be more effective than therapies which target capsule production.

Detection of anti-tat antibodies in CSF of individuals with HIV-associated neurocognitive disorders.

Despite major advances in the development of antiretroviral therapies, currently available treatments have no effect on the production of HIV-Tat protein once the proviral DNA is formed. Tat is a highly neurotoxic and neuroinflammatory protein, but its effects may be modulated by antibody responses against it. We developed an indirect enzyme-linked immunosorbent assay and measured anti-Tat antibody titers in CSF of a well characterized cohort of 52 HIV-infected and 13 control individuals. We successfully measured anti-Tat antibodies in CSF of HIV-infected individuals with excellent sensitivity and specificity, spanning a broad range of detection from 10,000 to over 100,000 relative light units. We analyzed them for relationship to cognitive function, CD4 cell counts, and HIV viral load. Anti-Tat antibody levels were higher in those without neurocognitive dysfunction than in those with HIV-associated neurocognitive dysfunction (HAND) and in individuals with lower CD4 cell counts and higher viral loads. We provide details of an assay which may have diagnostic, prognostic, or therapeutic implications for patients with HAND. Active viral replication may be needed to drive the immune response against Tat protein, but this robust immune response against the protein may be neuroprotective.

Burden of neuroinfectious diseases on the neurology service in a tertiary care center.

BACKGROUND: Neurologic infections have the potential to cause death and suffering. These disorders often go unrecognized or are misdiagnosed. There has yet not been a census of neurologic infections conducted in a hospital setting. We aimed to determine the burden of neurologic infections on the neurology service in a tertiary care center and identify challenges in the diagnosis and treatment of these infections. METHODS: We reviewed retrospectively all inpatients diagnosed with any neuroinfectious disease evaluated at Johns Hopkins Medical Institutions between October 2004 and December 2005. We recorded information on hospital admission, clinical features, microbiologic analysis, neuroimaging, EEG, pathology, treatment, and outcome. RESULTS: A total of 116 of 4,225 patients admitted to or consulted on by the neurology service were identified. Eighty percent of patients were aged between 18 and 65 years. Fifty-two patients were immunocompromised, of which 28 patients had HIV infection. Overall, 86 microbiologic agents were identified in 80 patients. The commonest causes were viral, followed by bacterial and fungal infections. However, 31% of patients remained without an identifiable microbiologic etiology. Hospitalization periods were long, with 43% of patients staying beyond 2 weeks. There was significant morbidity: 28% of patients required rehabilitation or long-term care, and 12% died. CONCLUSIONS: Neurologic infections have a major socioeconomic impact because they result in prolonged hospitalizations, expensive diagnostic tests and treatments, and long-term debilitation or death in young patients. Though potentially curable conditions, the burden of undiagnosed infections remains high.

Interaction of HIV Tat and matrix metalloproteinase in HIV neuropathogenesis: a new host defense mechanism.

Tat, the HIV transactivating protein, and matrix metalloproteinases (MMPs), a family of extracellular matrix (ECM) endopeptidases, have been implicated in the pathogenesis of HIV-associated dementia. However, the possibility that MMPs interact with viral proteins has remained unexplored. We therefore treated mixed human fetal neuronal cultures with recombinant Tat and select MMPs. Neurotoxicity was determined by measuring mitochondrial membrane potential and neuronal cell death. Previous studies have shown that Tat and MMP independently cause neurotoxicity. Surprisingly, we found the combination of Tat and MMP produced significant attenuation of neurotoxicity. To determine whether there was a physical interaction between Tat and MMP, we used protein electrophoresis and Western blot techniques, and found that MMP-1 can degrade Tat. This effect was blocked by MMP inhibitors. Furthermore, MMP-1 decreased Tat-mediated transactivation of the HIV long terminal repeat region, and this functionality was restored when MMP-1 activity was inhibited. These results suggest that the decrease in Tat-induced neurotoxicity and HIV transactivation is due to Tat's enzymatic cleavage by MMP-1. The direct interaction of human MMPs with viral proteins has now been demonstrated, with resultant modulation of Tat-mediated neurotoxicity and transactivation. This study elucidates a unique viral-host interaction that may serve as an innate host defense mechanism.

CADASIL: the dermatologic diagnosis of a neurologic disease. Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an increasingly recognized neurologic disease characterized by pathognomonic changes to the small vessels, particularly in the brain and skin. Although much has recently been written about this disease in the neuropathology literature, to our knowledge nothing has appeared in the dermatology literature. We wish to call attention to the unique role dermatologists and dermatopathologists can play in the diagnosis of this disease. We review the condition's clinical, histologic, and ultrastructural features.

Cleavage of substrates with mismatched nucleotides by Flap endonuclease-1. Implications for mammalian Okazaki fragment processing.

Flap endonuclease-1 (FEN1) is proposed to participate in removal of the initiator RNA of mammalian Okazaki fragments by two pathways. In one pathway, RNase HI removes most of the RNA, leaving a single ribonucleotide adjacent to the DNA. FEN1 removes this ribonucleotide exonucleolytically. In the other pathway, FEN1 removes the entire primer endonucleolytically after displacement of the 5'-end region of the Okazaki fragment. Cleavage would occur beyond the RNA, a short distance into the DNA. The initiator RNA and an adjacent short region of DNA are synthesized by DNA polymerase alpha/primase. Because the fidelity of DNA polymerase alpha is lower than that of the DNA polymerases that complete DNA extension, mismatches occur relatively frequently near the 5'-ends of Okazaki fragments. We have examined the ability of FEN1 to repair such errors. Results show that mismatched bases up to 15 nucleotides from the 5'-end of an annealed DNA strand change the pattern of FEN1 cleavage. Instead of removing terminal nucleotides sequentially, FEN1 appears to cleave a portion of the mismatched strand endonucleolytically. We propose that a mismatch destabilizes the helical structure over a nearby area. This allows FEN1 to cleave more efficiently, facilitating removal of the mismatch. If mismatches were not introduced during synthesis of the Okazaki fragment, helical disruption would not occur, nor would unnecessary degradation of the 5'-end of the fragment.

Processing of an HIV replication intermediate by the human DNA replication enzyme FEN1.

The role of human FEN1 (flap endonuclease-1), an RTH1 (RAD two homolog-1) class nuclease, in the replication of human immunodeficiency virus (HIV) type 1 has been examined using model substrates. FEN1 is able to endonucleolytically cleave a primer annealed to a template, but with a 5'-unannealed tail. The HIV (+)-strand is synthesized as two discontinuous segments, with the upstream segment displacing the downstream segment to form a central (+)-strand overlap. Given a substrate with the exact HIV nucleotide sequence, FEN1 was able to remove the overlap. After extension of the upstream primer with DNA polymerase epsilon, human DNA ligase I was able to complete the continuous double strand as would occur for an integrated provirus. FEN1 may represent a target for new therapeutic interventions.

Operation Pacific Haven: humanitarian medical support for Kurdish evacuees.

This article reviews the medical aspects of the humanitarian assistance mission Joint Task Force Operation Pacific Haven from September 1996 to April 1997. It reviews the effectiveness of the deployable medical units used to support the medical screening, treatment, and processing of more than 6,600 Kurdish evacuees applying for political asylum in the United States. The distinct cultural mores and language barriers of the Kurdish population made the provision of even basic medical care a challenge. Designed for combat service support, these deployable medical units were successful in the performance of the comprehensive public health and humanitarian assistance medical support mission because of the support of two on-island military treatment facilities. In short, for military medicine to successfully conduct humanitarian assistance and/or disaster relief missions, deployable medical units need to be designed, equipped, staffed, and trained to perform these operations.

Creation and removal of embedded ribonucleotides in chromosomal DNA during mammalian Okazaki fragment processing.

Mammalian RNase HI has been shown to specifically cleave the initiator RNA of Okazaki fragments at the RNA-DNA junction, leaving a single ribonucleotide attached to the 5'-end of the downstream DNA segment. This monoribonucleotide can then be removed by the mammalian 5'- to 3'-exo-/endonuclease, a RAD2 homolog-1 (RTH-1) class nuclease, also known as flap endonuclease-1 (FEN-1). Although FEN-1/RTH-1 nuclease often requires an upstream primer for efficient activity, the presence of an upstream primer is usually inhibitory or neutral for removal of this 5'-monoribonucleotide. Using model Okazaki fragment substrates, we found that DNA ligase I can seal a 5'-monoribonucleotide into DNA. When both ligase and FEN-1/RTH-1 were present simultaneously, some of the 5'-monoribonucleotides were ligated into DNA, while others were released. Thus, a 5'-monoribonucleotide, particularly one that is made resistant to FEN-1/RTH-1-directed cleavage by extension of an inhibitory upstream primer, can be ligated into the chromosome, despite the presence of FEN-1/RTH-1 nuclease. DNA ligase I was able to seal different monoribonucleotides into the DNA for all substrates tested, with an efficiency of 1-13% that of ligating DNA. These embedded monoribonucleotides can be removed by the combined action of RNase HI, cutting on the 5'-side, and FEN-1/RTH-1 nuclease, cleaving on the 3'-side. After FEN-1/RTH-1 action and extension by polymerization, DNA ligase I can join the entirely DNA strands to complete repair.

Calf RTH-1 nuclease can remove the initiator RNAs of Okazaki fragments by endonuclease activity.

In eukaryotes, the endonucleolytic activity of the calf RTH-1 class 5'- to 3'-exo/endonuclease can function without RNase H1 to remove initiator RNA from Okazaki fragments. Cleavage requires that the RNA be displaced to form an unannealed single-stranded 5'-tail or flap structure. On substrates with RNA-initiated primers, DNA oligomers that competed with the RNA for template binding simulated strand displacement synthesis from an upstream Okazaki fragment. This allowed cutting of displaced RNA segments by RTH-1 nuclease. Requirements for the reaction also were examined on substrates in which the tail was unannealed because it was intentionally mispaired. On both types of substrate, the nuclease slides over the RNA region from the 5'-end and cleaves at the beginning of the annealed region, irrespective of whether ribo- or deoxyribonucleotides are at the cleavage site. Presence of a triphosphate or a 7-methyl 3'G5'ppp5' G cap structure at the 5'-end of the RNA does not affect cleavage. The previously reported stimulation of the nuclease by an upstream primer was not always observed, suggesting that not every site in the downstream Okazaki fragment is equally susceptible to cleavage during displacement synthesis in vivo. The biological role of the endonuclease activity of RTH-1 nuclease in Okazaki fragment processing is discussed.

Role of calf RTH-1 nuclease in removal of 5'-ribonucleotides during Okazaki fragment processing.

The role of the exonucleolytic activity of the calf 5' to 3' exo/endonuclease, a RAD2 homolog 1 (RTH-1) class nuclease, in lagging-strand DNA replication has been examined using model Okazaki fragment substrates. These substrates exemplify the situation in Okazaki fragment processing which occurs after the initiator RNA primer is cleaved off, and released intact, by calf RNase HI, leaving a single ribonucleotide at the 5' end of the RNA-DNA junction. This final RNA is then removed by the calf RTH-1 nuclease [Turchi et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9803-9807]. The cleavage specificity of calf RTH-1 nuclease for different junction ribonucleotides was compared. These were removed without the usual requirement of calf RTH-1 for an immediately adjacent upstream primer. In most cases, the presence of an upstream DNA or RNA primer, separated from the monoribonucleotide-DNA segment by either a nick or a gap, reduced the efficiency of removal of the monoribonucleotide compared to the removal seen with no upstream primer. Substrates in which the monoribonucleotide-DNA segment had been replaced by an oligomer of the same sequence but consisting entirely of DNA also exhibited upstream primer inhibition. Results with various sequences indicated that the upstream primer is generally inhibitory for ribonucleotide removal but is sometimes neutral. For deoxynucleotide removal it could be stimulatory, neutral, or inhibitory. Possible reasons for the unexpected lack of upstream primer dependence have been explored. The ratio of RNase HI to RTH-1 was also shown to be critical for both enzymes to work together efficiently. These results suggest that regions of upstream primer inhibition within the genome may play a role in determining the mechanism by which mammalian Okazaki fragments are processed.

Photoimmunotherapy and biodistribution with an OC125-chlorin immunoconjugate in an in vivo murine ovarian cancer model.

Photodynamic therapy (PDT) is an experimental approach to the treatment of neoplasms in which photosensitisers (PSs) accumulated in malignant tissues are photoactivated with appropriate wavelengths of light. The target specificity of PSs may be improved by linking them with carrier macromolecules such as monoclonal antibodies (MAbs). OC125 is a murine MAb that recognises the antigen CA 125, which is expressed on 80% of non-mucinous ovarian tumours. A chlorin derivative conjugated to OC125 was shown to be selectively phototoxic to ovarian cancer and other CA 125-positive cells in vitro and ex vivo. We now report in vivo studies using an ascitic Balb/c nude mouse ovarian cancer model. Ascites was induced by intraperitoneal injection of cells from the human ovarian cancer cell line NIH:OVCAR3. Six weeks after injection, when the animals had developed ascites, biodistribution studies were carried out by injecting the immunoconjugate (IC) or free PS intraperitoneally and sacrificing the animals at 3, 6, 12, 24, 48, 72 and 168 h later. The PS was quantitated by extraction and fluorescence spectroscopy. For both the IC and free PS, peak tumour concentrations were reached at 24 h; however, the absolute concentrations for the IC were always higher (2- to 3-fold) than the free PS. Tumour to non-tumour ratios at 24 h for the IC were 6.8 for blood, 6.5 for liver, 7.2 for kidney, 5.7 for skin and 3.5 for intestine. Evaluation of viable tumour cells in ascites following in vivo PDT with a single light exposure demonstrated a dose-dependent relationship with fluence and IC concentration. However, there was significant treatment-related toxicity at all fluences. With multiple low-dose treatments, the percentage of viable tumour cells was also significantly reduced and there were no treatment-related deaths. These data suggest that, while photoimmunotherapy remains promising as a new treatment modality for ovarian cancers, careful quantitative dosimetry of both IC and light may need to be combined with multiple treatments (as with radiation therapy and chemotherapy) to control malignant disease yet maintain acceptable toxicity in vivo.

Medical success in El Salvador.

American medicine is respected worldwide, and our ability to respond with aid is beyond that of any nation. While we cannot administer aid indiscriminately, our ability to provide swift, effective humanitarian aid is one way in which we can demonstrate that we are truly relevant in the Third World. Recent United States experience in El Salvador proves this point. In 1983, when the Army was sending the first medical mobile training team to El Salvador, the mortality rate of wounded Salvadorian soldiers was 45%. As a result of U.S. military medical assistance, over the past four years the mortality rate of the wounded decreased to 5%. Activities of the Army Medical Department in El Salvador are an excellent example of efficient use of military medicine in low intensity conflict.