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HIV-1 subtype B virus is the most prevalent subtype in Puerto Rico (PR), accounting for about 90% of infection in the island. Recently, other subtypes and circulating recombinant forms (CRFs), including F(12_BF), A (01_BF), and CRF-39 BF-like, have been identified. The purpose of this study is to assess the distribution of drug resistance mutations and subtypes in PR. A total of 846 nucleotide sequences from the period comprising 2013 through 2017 were obtained from our “HIV Genotyping” test file. Phylogenetic and molecular epidemiology analyses were performed to evaluate the evolutionary dynamics and prevalence of drug resistance mutations. According to our results, we detected a decrease in the prevalence of protease inhibitor, nucleoside reverse transcriptase inhibitor (NRTI), and non-NRTI (NNRTI) resistance mutations over time. In addition, we also detected recombinant forms and, for the first time, identified subtypes C, D, and CRF-24BG in PR. Recent studies suggest that non-subtypes B are associated with a high risk of treatment failure and disease progression. The constant monitoring of viral evolution and drug resistance mutation dynamics is important to establish appropriate efforts for controlling viral expansion.

Schwannoma is a benign peripheral nerve sheath tumor originating from Schwann cells. Most intracranial schwannomas arise from vestibular nerve and schwannoma in the suprasellar region is extremely rare. A 64-year-old man presented with walking disturbance and blurred vision for three months. Lateral hemianopsia in the left eye and brachybasia were observed. Magnetic resonance imaging revealed a suprasellar tumor with strong contrast enhancement associated with communicating hydrocephalus. The cerebrospinal fluid tap test improved gait disturbance. Hypothalamic stimulation test revealed hypo-reaction of GH, FSH and LH. After ventriculo-peritoneal shunting, the tumor was totally removed via a bilateral front-basal approach with a clinical diagnosis of craniopharyngioma. No adhesion was observed between the tumor and surrounding structures such as meninges and brain. The histopathological diagnosis was schwannoma. Here we report a case of suprasellar schwannoma associated with communicating hydrocephalus that has not ever been previously reported, with special reference to its pathogenesis.

Alcohol-related disorders (ARD) are highly prevalent among Latin American-Caribbean countries. Mental disorders are common comorbidities in individuals with ARD. However, the etiology of the association between ARD and mental disorders remains unclear. We examined the association of inflammatory cytokines, microbiome, and other biomakers with measures of depression, social anxiety, and executive functions. We observed a significant increase in cytokine and chemokine expression levels in saliva and plasma in the alcohol group (AG) samples. Also, the salivary bacterial composition in the AG revealed an abundance of Prevotella. Depression symptomatology was markedly higher in the AG, but social anxiety levels were negligible. AG also exhibited executive dysfunctions, which negatively correlated with increased plasma levels of pro-inflammatory cytokines and increased salivary concentrations of Prevotella bacteria. Our study suggests that chronic alcohol use correlates with executive dysfunction, immune system dysregulation, and dysbiosis of the salivary microbiota. Additional studies are needed to understand the role of the microbiome and inflammation in alcohol use and mental comorbidities.

Puerto Rico has one of the highest diabetes rates in the USA and the prevalence of clinically diagnosed diabetes increased almost 5-folds between the years 2000 and 2004; and...

Tobacco use has been implicated as an immunomodulator in the oral cavity and contributes to the development of oral cancer. In the present study, we investigated the effects of cigarette smoking on bacterial diversity and host responses compared to healthy nonsmoking controls. Saliva samples were collected from eighteen smokers and sixteen nonsmoking individuals by passive drool. The 16S rRNA gene was used to characterize the salivary microbiome by using the Illumina MiSeq platform. Cytokine and chemokine expression analyses were performed to evaluate the host response. Significant differences in cytokine and chemokine expression levels of MDC, IL-10, IL-5, IL-2, IL-4, IL-7, adrenocorticotropic hormone (ACTH), insulin, and leptin were observed between smokers and nonsmokers. Taxonomic analyses revealed differences between the two groups, and some bacterial genera associated with the smokers group had correlations with hormones and cytokines identified as statistically different between smokers and nonsmokers. These factors have been associated with inflammation and carcinogenesis in the oral cavity. The data obtained may aid in the identification of the interactions between the salivary microbiome, host inflammatory responses, and metabolism in smokers.

The implementation of antiretroviral treatment combined with the monitoring of drug resistance mutations improves the quality of life of HIV-1 positive patients. The drug resistance mutation patterns and viral genotypes are currently analyzed by DNA sequencing of the virus in the plasma of patients. However, the virus compartmentalizes, and different T cell subsets may harbor distinct viral subsets. In this study, we compared the patterns of HIV distribution in cell-free (blood plasma) and cell-associated viruses (peripheral blood mononuclear cells, PBMCs) derived from ART-treated patients by using Sanger sequencing- and Next-Generation sequencing-based HIV assay. CD4+CD45RA−RO+ memory T-cells were isolated from PBMCs using a BD FACSAria instrument. HIV pol (protease and reverse transcriptase) was RT-PCR or PCR amplified from the plasma and the T-cell subset, respectively. Sequences were obtained using Sanger sequencing and Next-Generation Sequencing (NGS). Sanger sequences were aligned and edited using RECall software (beta v3.03). The Stanford HIV database was used to evaluate drug resistance mutations. Illumina MiSeq platform and HyDRA Web were used to generate and analyze NGS data, respectively. Our results show a high correlation between Sanger sequencing and NGS results. However, some major and minor drug resistance mutations were only observed by NGS, albeit at different frequencies. Analysis of low-frequency drugs resistance mutations and virus distribution in the blood compartments may provide information to allow a more sustainable response to therapy and better disease management.

Our long-term goal is to discover the combination of host parameters that help some HIV-infected individuals to resist progression to AIDS. In this study, we examined antibody responses using multiple samples obtained from a cohort of Long-Term Non-Progressors (LTNPs). Our hypothesis is that antibody responses to variable regions of the HIV-1 envelope glycoprotein are involved in reducing the viral load associated with LTNPs and that these specific immune responses influence susceptibility to disease progression. Multiple plasma samples were obtained from patients identified as LTNPs with the objective of characterizing humoral immune response directed to the five hypervariable regions of the envelope glycoprotein. Antibody binding was tested against peptides representing the five hypervariable regions of gp120, as well as against analog peptides representing different isolates of HIV-1 and against recombinant envelope glycoprotein. LTNPs have specific antibodies to the hypervariable regions of the envelope glycoprotein and develop different patterns of antibody recognition to variable epitopes of envelope glycoprotein. These antibodies can be detected using HIV peptides as capture antigens.

In Japan, the number of hemodialysis patients increases every year, along with the average age of this patient population. Further, certain complications of hemodialysis make the care of traumatic head injury(THI)patients particularly difficult.This study was aimed at investigating the occurrence of and risk factors for post-traumatic seizures in hemodialysis patients with a history of THI, and determining patient outcomes.Subjects were selected from patients who were admitted to Yaizu Municipal Hospital in Shizuoka, Japan for traumatic intracranial hemorrhage(TICH). Retrospective medical histories of TICH patients who were and were not receiving hemodialysis were reviewed to investigate the risk factors for seizures and to determine patient outcomes.We identified 18 THI patients on hemodialysis and 86 THI patients not on hemodialysis treatment. We determined that predictive factors of post-traumatic seizure include:current hemodialysis treatment, enlargement of an existing hematoma, and an acute subdural hematoma. Moreover, 66.7% of seizures in our dialysis patients occurred during hemodialysis. Our data also suggest that Glasgow Coma Scale(GCS)scores on admission are a predictive factor for patient outcomes following discharge.Current hemodialysis treatment, enlargement of an existing hematoma, and an acute subdural hematoma are predictive factors of seizure occurrence in THI patients. As post-traumatic seizures triggered unfavorable outcomes in some dialysis patients, it is important to create appropriate plans for preventing dialysis disequilibrium syndrome that may lead to seizures in TICH/TIH patients on hemodialysis. We also determined that a low GCS score upon admission is a significant predictor of unfavorable outcomes.

Background HIV-1 drug resistance in treatment-naive patients has a significant impact on the individual patient as well as implications for the wider population. These effects are amplified in the context of resource-limited settings, which are rapidly expanding access to antiretroviral therapy. Methods This cross-sectional survey at a single treatment site in Kingston, Jamaica was designed to identify the prevalence of HIV-1 drug-resistant mutations in chronically infected, treatment-naive patients. Mutations were identified using the Stanford HIV database algorithm and the World Health Organization (WHO) HIV Drug Resistance (HIVDR) surveillance mutations. Results The inclusion of 103 cases in the study resulted in 79 (76.6%) amplifiable samples. Genotype analysis revealed that 12.6% (95% CI 5.3, 19.9) were identified as having clinically significant mutations, while 10.1% (95% CI 3.5, 16.7) had WHO HIVDR surveillance mutations. Conclusions According to the WHO standard, this study population has a moderate level of HIVDR in treatment-naive patients and strongly implies the need to introduce HIVDR surveillance in Jamaica.

Memory CD4 T cells are the primary targets of HIV-1 infection, which then subsequently spreads to other T lymphocyte subsets. Antiretroviral therapy (ART) alters the pattern of HIV-1 distribution. Blood samples were collected from ART-naïve or -experienced HIV-1 patients, and the memory and naïve subsets of CD4(+) and CD8(+) T lymphocytes, respectively, were isolated by cell sorting. DNA was extracted and the HIV-1 env C2/V3 region PCR amplified. Amplicons were cloned and sequenced, and genetic relatedness among different HIV-1 compartments was determined by the phylogenetic analysis of clonal sequences. The viral V3 sequence of HIV-1 in each compartment was analyzed by using webPSSM to determine CCR5 or CXCR4 coreceptor binding property of the virus. The direction of viral migration among involved compartments was determined by using the MacClade program. In ART-naïve patients, HIV-1 was generally confined to the memory CD4 T (mT4) cell compartment, even though in a few cases, naïve CD4 T (nT4) cells were also infected. When this occurred, the HIV-1 gene migrated from nT4 to mT4. In contrast, HIV-1 was detected in nT4 and mT4 as well as in the memory CD8 T (mT8) compartments of ART-experienced patients. However, no clear pattern of directional HIV-1 gene flow among the compartments could be determined because of the small sample size. All HIV-1-infected T cell compartments housed the virus that used either CCR5 or CXCR4 as the coreceptor.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), persists within infected macrophages for long periods of time in a metabolically inactive but reversible state known as dormancy. Since the majority of adult pulmonary TB is caused by the reactivation of persistent Mtb, novel vaccines to protect against disease reactivation and novel biomarkers to provide the basis of new diagnosis of latent infection are urgently needed. To meet this demand, we assessed the immunogenicity of 32 latency-associated proteins in DNA-vaccinated mice. By using a Helios gene gun, BALB/c and C57BL/6 mice were vaccinated with plasmids carrying DNAs for latency-associated antigens encoded by the DosR regulon. Of the 32 antigens tested, 12 induced antigen-specific T-cell responses in vaccinated BALB/c mice and 9 induced responses in C57BL/6 mice. Five antigens (Rv1998c, Rv2031c, Rv2032, Rv2623, and Rv3132c) induced T-cell responses in both mice strains. In addition, at least 12 and 3 antigens induced antigen-specific antibody production in vaccinated BALB/c and C57BL/6 mice, respectively. Of these, 3 antigens (Rv2029c, Rv2626c, and Rv3132c) induced strong antibody production in both mice strains. These results might be applicable for the future development of a novel vaccine and biomarkers for latent TB infection, although further analyses in human blood samples are necessary.

Germline mutations in the p53 tumor suppressor gene have been identified in patients with Li-Fraumeni syndrome (LFS) and patients with Li-Fraumeni-like syndrome (LFL). However, to date, germline p53 mutations in patients not fulfilling the criteria of LFS or LFL have been reported only very rarely. In our study, a novel germline c.584T>C (p.Ile195Thr) mutation of the p53 gene was found in a 21-year-old male with a glioblastoma and colon cancer. He had no family history of cancer within second-degree relatives, and loss of the wild-type p53 allele and overexpression of p53 protein were observed in both tumors. Functional analyses revealed transactivation and growth suppressive function activities of the Thr195-type p53 to be impaired. These results suggest germline p53 mutations to possibly be responsible for a subset of young adult patient with multiple malignant tumors, even those not meeting the clinical criteria for LFS or LFL.

In order to assess the extent of Xylazine (Xyz) injection in Puerto Rico, two waves of used-syringe collections were performed. In the first, syringes were gathered, anonymously and without additional information; in the second, a short interview, also anonymous, was administered. We found Xyz in 37.6% of the collected syringes; the majority of the Xyz-containing syringes came from ranching communities. Syringes containing Xyz more frequently also contained “speedball” than those without (90.6% and 66.7%, respectively). Self-reports of Xyz injection deviated markedly from actual detection: only 50% (self-described users) and 22% (self-described non-users) of the collected syringes contained the drug. With a high prevalence of skin ulcers (38.5% vs. 6.8%, p < 0.001), Xyz users were more likely to be in poor health compared to non-users. Surprisingly, though a higher percentage of Xyz users than non-users had college-level educations (23.1% vs. 5.5%), they were more likely to be homeless (64.1% vs. 37%).

The role of Human Papillomavirus (HPV) in colorectal carcinogenesis remains elusive. Based on the high incidence of HPV-associated malignancies among Puerto Rican Hispanics, this study aimed to assess the prevalence of HPV infection and viral integration in colorectal tissues in order to evaluate its putative role in colorectal cancer (CRC). In this case-control study, the prevalence of HPV infection in CRC (casesn= 45) and normal colon mucosa from cancer-free subjects (controlsn= 36) was assessed by a nested PCR strategy. HPV-16 genotyping was performed in HPV-positive tissues and the physical status of the HPV-16 genome was determined by E2 detection. HPV was detected in 19 of 45 (42.2%) CRC cases (mean age 61.1 ± 10.7 years, 24 males) and in 1 of 36 (2.8%) controls (mean age 60.9 ± 9.6 years, 24 males) with an OR = 25.58 (95% CI 3.21 to 203.49). HPV-16 was detected in 63.2% of the HPV-positive colorectal tumors; genome integration was observed in all HPV-16 positive cases. This is the first report showing the high prevalence of HPV infections in Caribbean Hispanic colorectal tumors. Despite evidence of HPV integration into the host genome, further mechanistic analysis examining HPV oncoprotein expression and the putative role of these oncoproteins in colorectal carcinogenesis is warranted.

The viral kinetics of HERV-K in HIV-1-infected patients receiving highly active antiretroviral therapy (HAART) is not unknown. HERV-K kinetic modeling may provide insight into factors altering the effectiveness of HAART in suppressing HIV-1 burden. We conducted a longitudinal study measuring the HERV-K RNA titers in four patients with successful HIV-1-suppressive HAART and in six patients undergoing HAART failure. HERV-K titers were usually undetectable in patients with successful HAART, and when detected, HERV-K titers remained below 5000 copies/ml. On the other hand, HERV-K RNA was consistently detected in patients who failed to respond to HAART before and after HIV-1 rebounds (p < 0.001). Elevated HERV-K RNA titers frequently preceded HIV-1 rebounds. These results suggest that HERV-K viral load may predict HIV-1 reactivation. HERV-K RNA testing might be clinically useful in predicting the onset of HIV-1 resistance due to suboptimal antiretroviral drug levels and/or poor adherence to treatment.

Antibodies to HERV-K antigens have been linked to HIV-1 infection and expression of HERV-K proteins generates T-cell cytotoxic responses in many cancers. HERV-K RNA and protein abundance was measured in HIV-1-infected and control cells. In vitro exposure of HIV-1 laboratory-adapted and primary isolates on U87MG cells increased the expression of HERV-K RNA in a dose-dependent manner. HERV-K RNA and protein burdens were significantly increased in HIV-1-producing H9 cell lines compared to H9 cells. The expression of HERV-K was synergistically increased in HIV-1-infected PBMCs after stimulation with PMA/ionomycin. Furthermore, the expression of HERV-K in PBMCs, and particularly in CD4(+) T cells, was higher in HIV-1 patients compared to control subjects. The expression of HERV-K might be related to HIV-1 pathogenesis and AIDS-associated cancers.

Approximately 8% of the human genome sequence is composed by human endogenous retroviruses (HERVs), most of which are defective. HERV-K(HML-2) is the youngest and most active family and has maintained some proviruses with intact open reading frames (ORFs) that code for viral proteins that may assemble into viral particles. Many HERV-K(HML-2) sequences are polymorphic in humans (present in some individuals but not in others) and probably many others may be unfixed (not inserted permanently in a specific chromosomal location of the human genome). In the present study HIV-1 and HCV-1-positive plasma samples were screened for the presence of HERV-K(HML-2) RNA in an RT-PCR using HERV-K pol specific primers. HERV-K(HML-2) viral RNA sequences were found almost universally in HIV-1(+) plasma samples (95.33%) but were rarely detected in HCV-1 patients (5.2%) or control subjects (7.69%). Other HERV-K(HML-2) viral segments of the RNA genome including gag, prt, and both env regions, surface (su), and transmembrane (tm) were amplified from HERV-K pol-positive plasma of HIV-1 patients. Type 1 and type 2 HERV-K(HML- 2) viral RNA genomes were found to coexist in the same plasma of HIV-1 patients. These results suggest the HERV-K(HML-2) viral particles are induced in HIV-1-infected individuals.

Viral components of the human endogenous retroviruses type K (HERV-K) have been largely detected in plasma from HIV-1 infected individuals. A Sybr Green Real-Time RT-PCR approach was optimized for detection and quantitation of HERV-K RNA titers in plasma samples using the iCycler technology. The method detected 1000 HERV-K RNA copies/mL of plasma sample. The Intra- and Inter-assay performance revealed a coefficient of variations that ranged from 0.2 to 2.46%, demonstrating accuracy and reproducibility. We quantified the HERV-K RNA load in 20 HIV-1 patients receiving highly active antiretroviral therapy (HAART). We found increased HERV-K RNA titers in patients with non-suppressive HAART (patients who may develop drug-resistance and/or received suboptimal therapeutic doses), compared to suppressive regimens (p < 0.001). HERV-K RNA was not detected in HCV-1 positive or seronegative controls. Sequencing of Real-Time RT-PCR products revealed particular HERV-K subtypes activated in the HIV-1 infection. The application of this assay could expand the understanding of the role of HERV-K in the HIV-1 infection and others pathological conditions.

Alcohol abuse constitutes a major cohort among HIV-infected individuals. The precise effect of alcohol addiction on HIV pathogenesis remains inconclusive, however. This study was designed to determine the effect of alcohol dependence on virus replication and CD4 profiles in simian immunodeficiency virus/simian-HIV-infected rhesus macaques. A group of 3 male Indian rhesus macaques was adapted to a self-drinking model of alcohol consumption, whereas another group of 3 macaques was provided a Nutrasweet solution. After 7 weeks of alcohol consumption, the alcohol-dependent animals along with controls were intravenously inoculated with a mixture of SHIV(KU), SHIV(89.6)P, and SIV/17E-Fr. These animals were followed for a period of 24 weeks for complete blood cell counts, CD4 cell profiles, and viral loads in the blood and cerebral compartments. The alcohol and control groups showed comparable peak viral loads in the blood. The plasma viral load in the alcohol group was 31- to 85-fold higher than that in the control group at weeks 18 through 24 after infection, however. The pattern of cerebrospinal fluid viral replication was also comparable during the acute phase; however, the virus continued to replicate in the brain of alcohol-dependent animals, whereas it became undetectable in the controls. The extent of CD4 cell loss in the alcohol group was significantly higher than that in the control animals at week 1 after infection.

We analyzed the viral C2–V4 envelope diversity, glycosylation patterns, and dS/dN ratios of plasma HIV-1 in an attempt to better understand the complex interaction between viral quasispecies and the host-selective pressures pre- and post-HAART. Phylogenetic analysis of the envelope gene of five patients revealed monophyletic clustering in patients with higher CD4+ T cell counts and sequence intermingling in those with lower CD4+ T cells in relation to the stage of HAART. Our analyses also showed clear shifts in N-linked glycosylation patterns in patients with higher CD4+ T cells, suggesting possible distinct immunological pressures pre- and post-HAART. The relative preponderance of synonymous/nonsynonymous changes in the envelope region suggested a positive selection in patients with higher CD4+ T cells, whereas lack of evidence for positive selection was found in the patients with lower CD4+ T cells. An exception to the last analysis occurred in the only patient who reached complete viral suppression, maybe due to drug pressure exerted over the pol gene that may obscure the immune pressure/selection at the envelope in this analysis. All these indications may suggest that even when HAART generates viral suppression, quasispecies evolve in the envelope gene probably resulting from host-selective pressure.

Six rhesus macaques were adapted to morphine dependence by injecting three doses of morphine (5 mg/kg of body weight) for a total of 20 weeks. These animals along with six control macaques were infected intravenously with mixture of simian-human immunodeficiency virus KU-1B (SHIV KU-1B ), SHIV 89.6P , and simian immunodeficiency virus 17E-Fr. Levels of circulating CD4 + T cells and viral loads in the plasma and the cerebrospinal fluid were monitored in these macaques for a period of 12 weeks. Both morphine and control groups showed precipitous loss of CD4 + T cells. However this loss was more prominent in the morphine group at week 2 ( P = 0.04). Again both morphine and control groups showed comparable peak plasma viral load at week 2, but the viral set points were higher in the morphine group than that in the control group. Likewise, the extent of virus replication in the cerebral compartment was more pronounced in the morphine group. These results provide a definitive evidence for a positive correlation between morphine and levels of viral replication.

There is increasing evidence that male or female genital tract represent a distinct replication compartment for human immunodeficiency virus type 1 (HIV-1) and that such compartments may serve as a virus reservoir. Forty-four paired plasma and vaginal samples from HIV-infected females undergoing HAART were collected to examine the viral responses to antiretroviral therapy and to assess the possible role of the vaginal tract as a reservoir for drug-resistant variants. Twenty-one females had detectable viral RNA both in plasma and vaginal fluid, whereas 14 females had detectable virus only in plasma. Twelve paired samples were used to analyze HIV-1 pol sequences for the presence of drug resistance-associated mutations. Nine of the twelve paired samples exhibited discordant drug resistance mutation patterns. The other three females showed identical drug resistance-associated mutations. However, further examination of protease and RT showed numerous non-drug-associated mutations that corresponded to predefined CTL epitopes. These non-drug-associated mutations were different between plasma and vaginal viruses, suggesting that evolution of HIV-1 was independent in these two compartments.

PINK1 was recently found to be associated with PARK6 as the causative gene. We performed mutation analysis in eight inbred families whose haplotypes link to the PARK6 region. We identified six pathogenic mutations (R246X, H271Q, E417G, L347P, and Q239X/R492X) in six unrelated families. All sites of mutations were novel, suggesting that PINK1 may be the second most common causative gene next to parkin in parkinsonism with the recessive mode of inheritance.

AlphaB-crystallin is known as a small heat shock protein with a cytoprotective function. This study was undertaken to investigate the relationship between alphaB-crystallin and changes seen in Alzheimer's disease. The distribution and immunohistochemical characteristics of alphaB-crystallin positive neurones in the cerebral cortices of 4 patients with Alzheimer's disease were examined. AlphaB-crystallin positive neurones were mainly distributed in the limbic and paralimbic regions, namely parahippocampal gyrus, fusiform gyrus, cingulate cortex, middle and superior frontal gyrus, and insular cortex, which corresponded to commonly affected regions in Alzheimer's disease. Moreover, such neurones were present predominantly in the III or V layer of the cerebral cortex. The number of alphaB-crystallin positive neurones increased in parallel with the neuronal loss. Logistic regression analysis revealed a significant relation between the density of alphaB-crystallin positive neurones and that of extracellular neurofibrillary tangles (NFTs), with a correlation coefficient (r) of 0.57 and P < 0.0001 in 14 regions of the cerebral cortex. In contrast, the relation was not statistically significant between the density of alphaB-crystallin positive neurones and that of classical senile plaques, diffuse plaques or intracellular NFTs. Modified Gallyas-Braak (GB) staining on alphaB-crystallin positive neurone demonstrated several patterns of the structures: faint GB positive structures in the swollen perikaryon with straight neurites, fine granules compressed and contorted into fuzzy bundles, intensely GB positive filamentous structures together with fine granules and very intensely GB positive ring-like NFTs in a swollen perikaryon with curved neurites. In positive neurones, the density of ring-like NFTs correlated with that of atrophic perikaryon, or bent neurites and a decrease in the immunoreactivity of alphaB-crystallin. These data suggest that a close relationship exists between the appearance of alphaB-crystallin in neurones, extracellular NFTs, and neurofibrillary formation in alphaB-crystallin positive neurones in Alzheimer brain.

Flow cytometric analysis of T cells from HIV+ and normal individuals activated for 15 hr showed that the percentage of cells producing interferon-γ (INFγ) was enhanced approximately threefold (39 compared to 14%) in the HIV+ CD8+ population. Activation modes, other than anti-CD3 with PMA, were ineffective, and in no case did the percentage of HIV+ CD4+ T cells show increased INFγ production over controls. Enhanced INFγ production was not induced by either anti-CD3 or PMA alone, or anti-CD3 or ConA with anti-CD28, or enhanced by N-acetylcysteine. In contrast to INFγ production, the percentage of CD4+ T cells producing interleukin-2 (Il-2) greatly exceeded that of the CD8+ T cells. The results from flow cytometry analyses of HIV+ CD8+ T cells was supported by quantitative analysis of INFγ mRNA (by PCR) and INFγ secretion by ELISA. These methods showed a sixfold and three- to fivefold increase, respectively, on a per cell basis. As HIV infection progresses, as shown by loss of CD4+ T cells, the proportion of CD8+ CD28− T cells increases, and it is this T cell subset that is responsible for 80% or more of the enhanced INFγ production. The enhanced INFγ in HIV+ patients derives from two factors: the increase in CD8+ CD28− cells to 70% and the percentage producing INFγ (60%, compared to 21% for CD8+ CD28+ cells). Our findings of a substantial increase in INFγ production in HIV infection arising from the increased number of CD8+ CD28− T cells are compatible with clinical studies which show elevated INFγ in HIV+ serum and INFγ producing CD8+ T cells dominating HIV+ lymph nodes. We also found a significantly decreased proliferative response of the HIV+-derived CD8+ T cell fraction with coactivator anti-CD-28, in contrast to PMA (with anti-CD3), which is probably a reflection of the diminished population of CD8+ CD28+ T cells in HIV+ donors compared to normal donors (30.7 compared to 67.9%).

In an autopsy case of sporadic rapidly progressive lower motor neuron disease (MND), Gallyas-positive argyrophilic and ubiquitinated filamentous intracytoplasmic inclusions were found in the neurons. Clinically, 7 months prior to death, a 68-year-old woman experienced a history of rapidly progressive muscle weakness of all four extremities and bulbar sign, without sensory and autonomic disturbance. Two months later, she became unable to stand or walk. Four months after onset, she needed respiratory support, and subsequently died due to cardiorespiratory arrest. Neuropathological examinations revealed neuronal loss and associated gliosis in the lower motor neurons, except for ocular motor nuclei, Clark’s column, and accessory cuneate nucleus, and tract degeneration was observed in the middle root zone of the posterior column and spinocerebellar tract. No Bunina bodies or Lewy body-like hyaline inclusions were found in the anterior horns. Gallyas-positive argyrophilic filamentous inclusions were found in the lower motor neurons and in nerve cells of the Clark’s column, intermediate zone, posterior horn and accessory cuneate nucleus. These were positive with anti-ubiquitin antibody but negative with anti-tau (tau-2 and AT8) and neurofilament antibodies. Electron microscopic examinations disclosed randomly arranged tubular-like filamentous profiles, with a diameter of 12–14 nm, sometimes with amorphous granules in the perikaryon. This is the first report on the Gallyas-positive argyrophilic and ubiquitinated filamentous inclusions in neurons in MND.

Autosomal recessive juvenile parkinsonism (AR-JP) is a distinct clinical and genetic entity characterized by selective degeneration of nigral dopaminergic neurons and young-onset parkinsonism with remarkable response to levodopa. Recently, we mapped the gene locus for AR-JP to chromosome 6q25.2-q27 by linkage analysis and we identified a novel large gene, Parkin, consisting of 12 exons from this region; mutations of this gene were found to be the cause of AR-JP in two families. Now we report results of extensive molecular analysis on 34 affected individuals from 18 unrelated families with AR-JP. We found four different homozygous intragenic deletional mutations, involving exons 3 to 4, exon 3, exon 4, and exon 5 in 10 families (17 affected individuals). In addition to the exonic deletions, we identified a novel one-base deletion involving exon 5 in two families (2 affected individuals). All mutations so far found were deletional types in which large exonic deletion accounted for 50% (17 of 34) and the one-base deletion accounted for 6% (2/34); in the remaining, no homozygous mutations were found in the coding regions. Our findings indicate that loss of function of the Parkin protein results in the clinical phenotype of AR-JP and that subregions between introns 2 and 5 of the Parkin gene are mutational hot spots.

Autosomal recessive juvenile parkinsonism (AR-JP) is a distinct clinical and genetic entity characterized by selective degeneration of nigral neurons. Recently, the parkin gene responsible for AR-JP has been identified. To date, we found two different deletional mutations including single and multiple exonic deletions. In the present study, we identified two types of point mutations (Thr240Arg and Gln311Stop) involving exons 6 and 8 in the parkin gene of the AR-JP patients from two Turkish families. This is the first report on point mutations for the parkin gene. Furthermore, the Thr240Arg mutation was located on a consensus sequence for the site of phosphorylation by casein kinase II. Identification of its mutation provides an important clue as to the role of the Parkin protein in degeneration of the substantia nigra in the brain of AR-JP patients.

To clarify the genetic mode and clinical characteristics of familial early-onset parkinsonism with diurnal fluctuation, we studied 43 patients from 22 families. The estimated segregation ratio (0.2963) and absence of gender preponderance indicated autosomal recessive inheritance. Clinical features included the average age at onset of 26.1 years, parkinsonism with marked diurnal fluctuation, remarkable effect of levodopa, dyskinesias, dystonia, hyperreflexia, absence of dementia, and a benign course; autonomic symptoms were only mild if present. Autopsy study in one of our patients disclosed neuronal loss without Lewy bodies and the presence of melanin-poor neurons in the substantia nigra. Linkage analysis on 16 families mapped the disease gene to chromosome 6q25.2-27.

SummaryParkinson disease (PD) is a common neurodegenerative condition associated with degeneration of dopaminergic neurons in the zona compacta of the substantia nigra. There is increasing evidence that genetic factors play a role in the etiology of PD, although genetic heterogeneity is likely. An autosomal dominant syndrome with many similarities to sporadic PD has been mapped to 4q21-22 in a large Italian pedigree and has been found to be due to mutation of the alpha-synuclein gene. However, this gene appears to account for only a minority of PD, and a susceptibility locus for autosomal dominant parkinsonism has recently been mapped, on 2p13. Autosomal recessive juvenile parkinsonism (JP), which shows marked clinical similarity to PD, maps to 6q25.2-q27. We found linkage to this region in a group of 15 families from four distinct ethnic backgrounds. A full genomic screen excluded other candidate regions. We have constructed a detailed genetic map of the linked region and have mapped the position of the manganese superoxide dismutase gene (SOD2). Recombination events restricted the JP locus to a 6.9-cM region and excluded SOD2. The apparent homozygosity for null alleles at D6S955 in one family suggested a deletion and finer localization of the JP locus.

Early onset parkinsonism with diurnal fluctuation (EPDF) is a dopa-responsive parkinsonism characterized by early onset and improvement of parkinsonian symptoms after sleep. A pathologic study of one case of EPDF showed selective neuronal degeneration in the zona compacta of the substantia nigra without Lewy body formation. To determine the disease locus for EPDF, the authors examined 53 members of 17 EPDF families and analyzed 151 meioses. Multipoint linkage analysis gave a peak lod score of 14.2 at 1.0 cM telomeric to D6S305 and placed the disease locus in the 17-cM interval between D6S437 and D6S253, which is exactly the same position mapped for autosomal recessive juvenile parkinsonism (ARJP; Mendelian inheritance in man 600116). The highest two-point lod score was obtained at D6S305 (10.13 at theta = 0). Haplotype analysis agreed with the result of multipoint analysis. A high proportion of nonconsanguinity (8/17 families), the absence of commonly shared haplotypes, and the widespread geographic distribution of the families' origins suggest that EPDF has multiple founder mutations. The authors concluded that EPDF and ARJP are allelic disorders and should be included in the spectrum of a single-clinicogenetic entity of chromosome 6q-linked parkinsonism. This disease could be characterized by a benign clinical course and relatively selective degeneration of nigral dopaminergic neurons with no Lewy body formation. To determine whether ARJP/EPDF constitutes a significant proportion of familial or sporadic forms of benign early onset parkinsonism will require further study.

Parkinson's disease is a common neurodegenerative disease with complex clinical features1. Autosomal recessive juvenile parkinsonism (AR-JP)2,3 maps to the long arm of chromosome 6 (6q25.2-q27) and is linked strongly to the markers D6S305 and D6S253 (ref. 4); the former is deleted in one Japanese AR-JP patient5. By positional cloning within this microdeletion, we have now isolated a complementary DNA clone of 2,960 base pairs with a 1,395-base-pair open reading frame, encoding a protein of 465 amino acids with moderate similarity to ubiquitin at the amino terminus and a RING-finger motif at the carboxy terminus. The gene spans more than 500 kilobases and has 12 exons, five of which (exons 3–7) are deleted in the patient. Four other AR-JP patients from three unrelated families have a deletion affecting exon 4 alone. A 4.5-kilobase transcript that is expressed in many human tissues but is abundant in the brain, including the substantia nigra, is shorter in brain tissue from one of the groups of exon-4-deleted patients. Mutations in the newly identified gene appear to be responsible for the pathogenesis of AR-JP, and we have therefore named the protein product ‘Parkin’.

CD8+ T-lymphocytes from HIV+ individuals contain short telomeres, a sign of cell senescence. To test our hypothesis that the cell type is functionally defective in the biochemical indices related to cell proliferation, we investigated the profiles of intracellularly generated H2O2 levels with or without PMA as well as immunoreactive catalase levels using flow cytometric method. We observed that, in HIV+ but not in HIV− individuals, the constitutively generated H2O2 level was significantly lower in CD8+ T-cells compared with CD4+ T-cells. Importantly, activated effector CD8+CD28− cells showed remarkably low H2O2 levels compared with CD8+CD28+ cells, and the latter in HIV+ individuals also showed low levels. A similar defect of CD8+ cells of HIV+ individuals was also seen with H2O2 levels stimulated with PMA in the presence of a catalase inhibitor. Furthermore, the immunoreactive catalase content was lower in CD8+ cells compared with CD4+ cells only in HIV+ individuals. These results suggest that CD8+ T-lymphocytes are functionally defective with the constitutively generated and PMA-elicited levels of H2O2 and the corresponding scavenger. Diminished immunocompetence of HIV+ individuals may be caused, in part, by the functional defect of CD8+ T-cells.

Background: Patients with Inflammatory Bowel Disease (IBD), most commonly Crohn’s disease (CD) or ulcerative colitis (UC), suffer from chronic intestinal inflammation of unknown etiology. Increased proinflammatory macrophages (M1) have been documented in tissue from patients with CD. Anti-inflammatory macrophages (M2) may play a role in UC given the preponderance of Th2 cytokines in this variant of IBD. Animal and clinical studies have shown that the probiotic VSL#3 can ameliorate signs and symptoms of IBD. Although animal data suggests a modulatory effect on macrophage phenotype, the effect of VSL#3 on human macrophages remains unknown.Objective: To determine the effect of the probiotic VSL#3 on the phenotype of polarized (M1/M2) and unpolarized (MΦ) human macrophages.Methods: Human monocyte-derived macrophages, generated by culturing monocytes with M-CSF, were left unpolarized or were polarized towards an M1 or an M2 phenotype by culture with LPS and IFN-γ or IL-4, respectively, and were then cultured in the presence or absence of VSL#3 for 3 days. Changes in macrophage morphology were assessed. Cytokine and chemokine levels in supernatants were determined by multiplex assay.Results: VSL#3 decreased the granuloma-like aggregates of M1 macrophages, increased fibroblast-like M2 macrophages, and decreased fibroblast-like MΦ macrophages. VSL#3 increased the secretion of IL-1β, IL-6, IL-10, and G-CSF by M1, M2, and MΦ macrophages. VSL#3 exposure maintained the proinflammatory phenotype of M1 macrophages, sustaining IL-12 secretion, increasing IL-23 secretion, and decreasing MDC secretion. Both VSL#3- treated M2 and MΦ macrophages secreted higher levels of anti-inflammatory and pro-healing factors such as IL-1Ra, IL-13, EGF, FGF-2, TGF-α, and VEGF, as well as proinflammatory cytokines, including IL-12 and TNF-α.Conclusion: Under our experimental conditions VSL#3 induced a mixed proinflammatory and anti-inflammatory phenotype in polarized and unpolarized macrophages. This differential effect could explain why patients with CD do not respond to probiotic therapy as well as patients with UC.

Objective: Depression is the most common psychiatric diagnosis in the HIV/AIDS population and represents a risk factor for disease progression. Since HIV-1 infection is characterized by immunologic and metabolic disturbances, we want to study the effects of depression on different components related to pro-inflammatory and oxidative stress markers. We hypothesize that depression will lead to increased pro-inflammatory cytokine levels and altered antioxidant/oxidant balance.Methods: We included males and females who were ≥21 years of age, whose HIV-1 sero-status was confirmed by Western Blot, and who were currently undergoing antiretroviral treatment. Patients completed the participation consent form, a socio-demographic survey, and the Patient Health Questionnaire-9 (PHQ-9) for depression assessment. We isolated the plasma from participants’ blood samples for viral load analysis (RT-PCR), T-cell counts (flow cytometry), and hematological parameters. A cytokine magnetic bead panel was used to measure interleukin-15 (IL-15), interferon gamma-induced protein 10 (IP-10), IL-12 and granulocyte colony-stimulating factor (G-CSF) levels. We also performed assays to determine the antioxidant activity of superoxide dismutase (SOD) and catalase and to measure the lipid peroxidation levels using malondialdehyde (MDA) and 8-isoprostane assays. Statistical comparisons and correlations at 5% level of significance were determined.Results: Our results show that subjects with mild/moderate to severe depression as assessed by PHQ-9 had a significantly decreased adherence to anti-retroviral treatment. Subjects with depression also had significantly lower levels of white blood cells (WBC) and platelets (PLT) than did the non-depressed group. The HIV+ subjects with depression had increased levels of IL-15, IP-10, IL-12 p40/p70 and G-CSF compared to their non-depressed counterparts. The latter had increased MDA and 8-isoprostane levels.Conclusions: Our results suggest that HIV+ subjects with depressive symptoms have higher levels of inflammation and altered oxidant/antioxidant balance. Although the groups were small, this study strengthens the hypothesis that alterations in cytokines are associated with the mechanisms underlying depression symptoms.

The human dopamine transporter (hDAT) is a member of the subfamily of monoamine transporters which show a common topological structure and possess significant amino acid sequence homology. We isolated and characterized the hDAT gene including about 1 kb of 5'-flanking region. The hDAT gene spans over 64 kb, consisting of 15 exons separated by 14 introns. The intron-exon structure of the hDAT gene is most similar to that of the human noradrenaline transporter (hNAT) gene. Promoter sequence analysis demonstrated a 'TATA'-less, 'CAT'-less and G+C-rich structure. Two E box and several Sp-1-binding sites exist in the promoter region. These structural features are similar to that of the human D1A dopamine receptor gene and the human monoamine oxidase A gene. The transcription start site was determined by both 5'-RACE and RNase protection assay. We determined the 5' end of the mRNA by identifying the 5'-terminal cap-G residue in 5'-RACE and RNase protection assay.

Among the modes of transmission available to the cytomegalovirus (CMV) is sexual transmission, primarily via semen. Both male-to-female (M-F) and male-to-male (M-M) sexual transmission significantly contribute toward the spread of CMV infections in the global population. Semen plays an important role in carrying the viral particle that invades the vaginal or rectal mucosa, thereby initiating viral replication. Both semen and seminal plasma (SP) can enhance HIV-1 infection in cell culture, and two amyloid fibrils, semen-derived enhancer of viral infection (SEVI) and amyloids derived from the semenogelins (SEM amyloids), have been identified as seminal factors sufficient to enhance HIV-1 infection (J. Munch et al., Cell 131: 1059–1071, 2007; N. R. Roan et al., Cell Host Microbe 10: 541–550, 2011; F. Arnold et al., J. Virol. 86:1244–1249, 2012). Whether SP, SEVI, or SEM amyloids can enhance other viral infections has not been extensively examined. In this study, we found that SP, SEVI, and SEM amyloids strongly enhance both human CMV (HCMV) and murine CMV infection in cell culture. SEVI and SEM amyloids increased infection rates by >10-fold, as determined by both flow cytometry and fluorescence microscopy. Viral replication was increased by 50- to 100-fold. Moreover, viral growth curve assays showed that SP, SEVI, and SEM amyloids sped up the kinetics of CMV replication such that the virus reached its replicative peak more quickly. Finally, we discovered that SEM amyloids and SEVI counteracted the effect of anti-gH in protecting against CMV infection. Collectively, the data suggest that semen enhances CMV infection through interactions between semen amyloid fibrils and viral particles, and these interactions may prevent HCMV from being neutralized by anti-gH antibody.

頭部MRIでみられる小血管病変に基づくと考えられる虚血性病変と脳血管障害の危険因子との相関を検討した.その結果, 脳室周囲病変ならびに皮質下白質部, 基底核部や脳幹部の小虚血病変の危険因子として加齢の重要性を再確認した.さらに, これらの小血管病変は症候性脳梗塞において, 無症候性脳梗塞に比較して, その程度がより高度であったことから, 無症候性脳梗塞にみられるこれらの病変は, 症候性脳梗塞の前段階を示唆していると考えられる.皮質下白質部と基底核部との小梗塞病変は, ともに, 加齢, 脳血管障害や高血圧の既往との間に関連があったことから, 小血管病変においては, 皮質動脈から分枝する髄質動脈領域と穿通枝動脈領域との間に共通した成因の存在を示唆した.小血管病変と総コレステロール, 中性脂肪やHDL-コレステロールとの問には有意の相関がなかった.したがって, 本研究の結果は, 小血管病変は大血管病変とは危険因子に相違があることを示唆した.

Development of a drug-resistant variant of HIV-1 has been one of the major concerns contributing to the transmission of the virus. A 40-year-old woman presented to the clinic with micosis and oral candidiasis. The subject was referred for HIV-1 diagnosis. Subsequent investigations revealed a very low CD4 T cell count (48 cell/microl blood) and high plasma HIV-1 RNA load (4.33 x 10(5) copy/ml). A 1.3-kb pol fragment was sequenced in virus collected from plasma and the vaginal compartment. Plasma virus had no mutation in reverse transcriptase and one mutation in protease (L63P). On the other hand vaginal virus contained L63P and M184V mutations in protease and reverse transcriptase, respectively. These mutations were accompanied by several other mutations in previously identified CTL epitopic regions of the two genes. In the absence of antiretroviral treatment, a drug-resistant mutant was thought to develop because of immune pressure. This is the first report describing the role of immune pressure in the development of a drug-resistant virus.

The cerebral cortex of patients with Alzheimer's disease (AD) or amyotrophic lateral sclerosis (ALS) may show low signal intensity on T2-weighted magnetic resonance images (MRI). Since these low intensity areas (LIA) are also often observed in aged patients with other diseases, we suspected that they might be a non-specific finding. We conducted a retrospective study of LIA in 139 patients with various diseases of the central and peripheral nervous systems, and evaluated their relationship to age and other MRI findings. Brain atrophy, ventricular dilatation, white matter lesions, and LIA were visually evaluated on axial images of the spin echo sequences obtained with a 1.5 tesla (T) system. We found that LIA appeared after age 50 and became more common with advancing age. Their presence correlated with brain atrophy and white matter lesions. They were most frequent in the motor cortex, followed by the occipital and sensory cortices. Their incidence in the motor cortex was significantly higher in patients with central nervous system diseases than in those with peripheral neuropathy. We conclude that LIA are common in old patients with various neurological diseases and suggest that the deposition of iron in the cerebral cortices causes their development.

We previously reported finding the RNA of a type K human endogenous retrovirus, HERV-K (HML-2), at high titers in the plasma of HIV-1-infected and cancer patients (R. Contreras-Galindo et al., J. Virol. 82:9329–9236, 2008.). The extent to which the HERV-K (HML-2) proviruses become activated and the nature of their activated viral RNAs remain important questions. Therefore, we amplified and sequenced the full-length RNA of the env gene of the type 1 and 2 HERV-K (HML-2) viruses collected from the plasma of seven HIV-1-infected patients over a period of 1 to 3 years and from five breast cancer patients in order to reconstruct the genetic evolution of these viruses. HERV-K (HML-2) RNA was found in plasma fractions of HIV-1 patients at a density of ∼1.16 g/ml that contained both immature and correctly processed HERV-K (HML-2) proteins and virus-like particles that were recognized by anti-HERV-K (HML-2) antibodies. RNA sequences from novel HERV-K (HML-2) proviruses were discovered, including K111, which is specifically active during HIV-1 infection. Viral RNA arose from complete proviruses and proviruses devoid of a 5′ long terminal repeat, suggesting that the expression of HERV-K (HML-2) RNA in these patients may involve sense and antisense transcription. In HIV-1-infected individuals, the HERV-K (HML-2) viral RNA showed evidence of frequent recombination, accumulation of synonymous rather than nonsynonymous mutations, and conserved N-glycosylation sites, suggesting that some of the HERV-K (HML-2) viral RNAs have undergone reverse transcription and are under purifying selection. In contrast, HERV-K (HML-2) RNA sequences found in the blood of breast cancer patients showed no evidence of recombination and exhibited only sporadic viral mutations. This study suggests that HERV-K (HML-2) is active in HIV-1-infected patients, and the resulting RNA message reveals previously undiscovered HERV-K (HML-2) genomic sequences.

Various brain tumors are components of familial cancer syndromes, and Li-Fraumeni syndrome and Li-Fraumeni-like syndromes are the most famous entities. A rare, sporadic occurrence of brain tumor in peculiarly young subjects, however, sometimes provides a clue to understanding of carcinogenesis due to germline mutations. In this section, two cases of glial tumors in subjects having germline mutation of TP53 are presented, and genetic etiology of brain tumor is discussed. The first case is a 41 year-old father of a fatal adrenocortical carcinoma case of a 4-years-old daughter, 16 years prior to the occurrence of astrocytoma of himself. The search for family history and previous clinical records in the four different community hospitals for the last three decades and the follow-ups of his family members disclosed cancer of urinary bladder, cancer of pancreas, hepatoblastoma, and thymoma in the relatives. Germline mutation E286A of TP53 was identified in the affected members. Another case is a 21-year-old male, without any family history of cancer, who suffered from brain tumor and colorectal cancer. An attending physician was sticky to find an etiology in this unusual occurrence of cancer. After several attempts in vain, an I195T germline mutation was identified and functional analysis was performed. These anecdotes highlight importance of genetic analysis in case of glial tumors in relatively young adult or in adolescence whether or not they have family history of cancer. The problems and strategies to find TP53 mutation carriers and to prevent or to delay the occurrence of the tumors in them are discussed.

Research into familial Parkinson's disease (PD) remained at a virtual standstill in Europe and the US for several decades until a re-challenge by Japanese neurologists regarding an autosomal recessive form of PD. In 1965, our research group at Nagoya University examined familial cases of early-onset parkinsonism characterized by autosomal recessive inheritance, diurnal fluctuation of symptoms (alleviation after sleep), foot dystonia, good response to medication, and benign course without dementia. An inborn error of metabolism in some dopamine-related pathway was suspected. The clinical study of four families with the disease, named as "early-onset parkinsonism with diurnal fluctuation (EPDF)", was published in Neurology in 1973. The pathological study of a case in 1993 revealed neuronal loss without Lewy bodies in the substantia nigra. Based on these clinical and pathological evidences, EPDF was defined as a distinct disease entity. Screening for the EPDF gene was started in 1994 in collaboration with Juntendo University. With the discovery of parkin gene in 1998, EPDF was designated as PARK2. Of our 16 families examined for gene analysis, 15 proved to be PARK2, and the remaining one, PARK6.