A human immunodeficiency virus type 1 (HIV) -seropositive, antiretroviral-naive patient presented with significant cognitive dysfunction. Neuropsychologic, neuroradiologic, immunologic, and virologic studies confirmed HIV-associated dementia (HAD). After 12 weeks of highly active antiretroviral therapy (HAART) with ibuprofen, dramatic improvements were demonstrated in neurologic function and were sustained for ú1 year. HIV-1 RNA in cerebrospinal fluid (CSF) decreased from 10 5 to 10 4 copies/mL after 4 weeks. After 20 weeks of therapy, plasma viremia decreased from 10 6 copies/mL to undetectable (õ96 copies/mL). Assays of neurotoxins (tumor necrosis factor-a, quinolinic acid, and nitric oxide) in plasma and CSF were considerably elevated at presentation and significantly decreased after therapy. Baseline plasma and CSF demonstrated neurotoxic activities in vitro, which also reduced markedly. These data, taken together, support the notion that HAD is a reversible metabolic encephalopathy fueled by viral replication. HAART used with nonsteroidal antiinflammatory agents leads to the suppression of inflammatory neurotoxins and can markedly improve neurologic function in HAD.Human immunodeficiency virus type 1 (HIV)-associated deproteins such as gp120, tat, and nef [18][19][20][21][22]. High levels of these macrophage-produced neurotoxic factors have been found mentia (HAD) is characterized by cognitive and motor impairment in 15%-20% of infected patients [1][2][3][4]. Neurologic defiin the brains and cerebrospinal fluid (CSF) of patients with HAD [23][24][25][26]. These factors produce an encephalopathy with cortical cits have been classified by the American Academy of Neurology, and an algorithm has been developed for mild disand subcortical neuronal damage [27] and induce a breakdown of the blood-brain barrier (BBB) [28, 29]. ease (minor cognitive/motor disorder) or more severe impairment (HAD) [1]. Dementia can occur with few neuropathologic HIV RNA levels in CSF correlate with the presence and severity of cognitive dysfunction [30 -33]. If a central nervous changes or with encephalitis characterized by multinucleated giant cells, astrocytosis, microglial nodules, myelin pallor, and system virus is both necessary and sufficient to induce neurologic impairment, therapy for HAD should of necessity target neuronal loss [5,6]. Of interest, clinical disease is not caused by active HIV replication in neural cells. The virus's target cells HIV. Inhibitors of HIV protease in combination with nucleoside analogue reverse transcriptase (RT) inhibitors can dramatiare mononuclear phagocytes (brain macrophages and microglia) [7,8]. Once infected, these cells become immunoactive and cally reduce levels of plasma viremia [34,35]. Unfortunately, many of these agents are highly plasma protein bound and have produce a variety of neurotoxic secretory products [9]. These include but are not limited to cytokines such as tumor necrosis limited BBB penetration [36]. Zidovudine remains the only drug to date with documented efficacy for H...

Newly synthesized major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum are thought to bind peptides of foreign and endogenous antigens. Several lines of evidence indicate that beta-2 microglobulin (beta 2m) and/or peptide ligand participate in the intracellular transport and surface expression of class I molecules, but the nature of their involvement is still unclear. Here we present evidence that culturing non-mutant cells (fibroblast, thymoma or mastocytoma) with a peptide ligand specific for the Ld class I molecule of the mouse leads to a dramatic (fourfold) and specific induction of Ld surface expression. Surprisingly, this peptide ligand-induced expression of Ld does not result in an increased intracellular association of Ld with beta 2m. These findings demonstrate that the previously reported decrease in surface expression of Ld results from its failure to be saturated with endogenous self-peptide ligands. This unique feature of Ld could also contribute to the fact that several virus-specific cytotoxic T cell responses have been found to be Ld-restricted.

We describe the use of fluorescent-labeled primers to analyze T-cell receptor gamma gene rearrangements (TCR gamma GR) using capillary electrophoresis in the ABI Prism 310 Genetic Analyzer. We also compare the performance with denaturing gradient gel electrophoresis (DGGE). In a single multiplex polymerase chain reaction (PCR) we amplified TCR gamma GR with primers for all known groups of variable region genes, and joining region genes described in lymphoid neoplasms. Ten reactive samples, followed by five cell lines and 25 tumor samples with 41 individual TCR gamma GR (due to many biallelic rearrangements) previously identified by DGGE, were analyzed to validate the technique. The capillary electrophoresis protocol has 92% concordance for both TCR clonal status (23 of 25) and 95% concordance in the number of individual TCR gamma GR (38 of 41) identified by DGGE. The reproducible sensitivity for detecting TCR gamma GR diluted in reactive lymphoid DNA is 2% in clinical applications. Discrimination of predominant rearrangements requires a minimum ratio of two times the height of the normal distribution of polyclonal peaks. Capillary electrophoresis can provide results within 60 minutes for each specimen after PCR is complete. Capillary electrophoresis provides a faster result than sequence-based separation methods and gives an archival electronic record. Fluorescent labeling allows the identification of both the variable and joining gene segments used in a TCR gamma GR. The effectiveness of capillary electrophoresis is similar to DGGE.

Immune reconstitution resulting from use of highly active antiretroviral therapy in patients infected with human immunodeficiency virus type 1 (HIV-1) has been associated with a significant decrease in infectious morbidity and with improved survival. Occasionally, patients with quiescent disease due to human cytomegalovirus or nontuberculous mycobacteria may experience paradoxical worsening due to "dysregulated" restitution of the immune system (that is, immune restoration disease [IRD]). Acquired immunodeficiency syndrome-related progressive multifocal leukoencephalopathy (PML) is uncommon and often improves with immune recovery. We describe 2 HIV-1-infected patients with PML that presented with paradoxical worsening after the patients had commenced active antiretroviral therapy. After they had a transient response to high-dose corticosteroid therapy, both patients died of progressive neurological deterioration. IRD in these patients with PML was unexpected and occurred soon after they had started receiving active antiretroviral therapy, during the period of improved antigen-specific T-helper cell function. Predictors of patients' proclivity for these adverse events are uncertain. Evaluation of targeted immunomodulatory therapy directed towards disease-specific IRD is critical and may play an important role in improved survival for patients who are at risk.

Two phenomena appear to distinguish the D region class I genes from those in the K region in the murine MHC: (a) haplotype disparity in the number of expressed D region class I molecules has been observed; and (b) clines of closely related D region class I molecules among and within mice of different H-2 haplotypes can be defined. Both of these observations have been based on serological and peptide mapping analyses of these molecules. Recent reports using molecular biological approaches have corroborated these findings. Since the mouse strain B10.AKM expresses multiple D region class I antigens, all of which are closely related to the prototypic Ld molecule, we investigated the Dq region of B10.AKM using molecular approaches. Three D region class I genes were isolated from genomic B10.AKM bacteriophage and cosmid libraries. Based on alignment of those genes with the BALB/c D region class I genes by analogous restriction endonuclease sites and by hybridization of one of those genes with a D4d gene-derived oligonucleotide probe, we have designated these genes as Dq, Lq, and D4q. As determined by DNA-mediated gene transfer to mouse L cells followed by serological analyses, the Dq and Lq genes encode previously characterized Dq region class I antigens. The nucleic acid sequence comparisons of the Dq and Lq genes demonstrated a higher level of homology with the Ld and Db genes than with other D region class I genes. In addition, CTL stimulated with a Dq, Lq, or Ld gene transfectant showed strong crossreactions with the other transfectants as targets, suggesting that the products of these genes are also functionally related. Thus, these studies suggest that the L molecule represents a prototypic structure shared by several D region gene products, and furthermore, the duplication of an Ld-like progenitor gene resulted in two Dq region class I genes, Dq and Lq. Unexpectedly, the sequences determined for the Dq and Lq genes are nearly identical to the sequences of two genes, A166 and A149, respectively, which were reported to encode the tumor-specific antigens; these novel class I genes were isolated from an H-2k fibrosarcoma, 1591. This raises the distinct possibility that these purported tumor-specific class I genes were introduced into this tumor by contamination.

Limited data exist regarding the distribution of gene segments used in T-cell receptor gamma gene rearrangements (TCR gamma GR) in T-cell lymphoproliferative disorders. The reported efficacy of TCR gamma GR protocols ranges from 60% to greater than 90%. Laboratories reporting a lower detection rate tend to use a limited set of primers. The goal of our study was to provide TCR gamma GR data to demonstrate the molecular biological basis for needing multiple primer sets targeting all gene segments. Sixty cases with a confirmed histological diagnosis of a T-cell lymphoproliferative disorder and TCR gamma GR were identified in our lymphoma registry from 1995 to 2001. DNA was obtained from fresh/frozen tissue, cell lysates, or paraffin-embedded tissue. Variable (V gamma) region gene segments were identified using denaturing gradient gel electrophoresis, which was used to select the cases in the study. Capillary electrophoresis using fluorescent-labeled joining (J gamma) region primers was performed to identify J gamma segments. Sixty cases contained a total of 98 TCR gamma GR, as some cases have more than one rearrangement. The most frequent gene segment combination involved the V gamma 1-8 and J gamma 1/2 segments. If a single primer set directed at these two segments were used for clinical diagnosis, that pair of primers would only diagnose 67% of cases as positive for TCR gamma GR. Our gene segment distribution data emphasize the importance of using a comprehensive set of V gamma and J gamma primers for an optimal detection rate of TCR gamma GR. Protocols with limited numbers of primers should be reconsidered.

Inbred mouse strains carrying spontaneous mutations in class I genes have been extremely informative in studies ofthe genetic mehanisms generating polymorphism in the major histocompatibility gene complex. In this report, we determine the molecular basis of the spontaneous loss mutation in BALB/c-H-2dm2 mice, which. were previously shown not to express Ld antigens while maintaining normal expression of two other class I antigens, Kd and Dd. We show BALB/c-H-2dm2 mice do not transcribe detectable levels ofUd mRNA, indicating they do not produce a truncated Ld molecule as previously reported.

The class I genes of the murine MHC encode glycoproteins that are noncovalently associated with a2-microglobulin (02m)t . These class I genes are located in four regions of the MHC, the K, D, Q, and TL regions. The classical transplantation antigens of the K and D regions function by presenting endogenous peptides derived from processed self or foreign proteins to the TCR (1), while the function ofthe Q and TL molecules is not known. In addition, the K and D region molecules display a high level of polymorphism, whereas few alleles have been demonstrated for the Qand TL class I genes (2). The high level ofpolymorphism in the transplantation antigens is due to amino acid substitutions concentrated in or near the peptide binding groove, as shown by recent crystallization data (3). Analyses of the K and D region class I genes from wild-type and mutant genes suggest that gene conversion-like events may play a major role in the generation of diversity (4). This high level of diversity is thought to increase the immune repertoire of individual members as well as the species as a whole.Although the number of K region class I genes and products has been found to be constant in different haplotypes, the number ofD region class I genes and products varies in different haplotypes (5, 6). Haplotype disparity involving the D region genes has been demonstrated in both inbred laboratory and wild-derived mouse strains using serological and peptide mapping analyses (7-11) . This disparity has been investigated at the molecular level in four haplotypes, b, k, p, and d (6, 12-14) . Of these haplotypes, all have a single D region gene, except the d haplotype, which contains five D region class I genes (12) . However, it is not clear whether there are only these two D region organizations. Evidence suggesting that this region represents a dynamic state of expansion and contraction is provided by the dml and dm2 mutant mouse strains. Molecular studies of these mutants revealed deletions of several