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Biomedical subjects

Robert F Garry

Publications and source records attributed to Robert F Garry.

At least 19 recordsLinked to original sources
PubMed2007

Aluminum adjuvant linked to Gulf War illness induces motor neuron death in mice.

Gulf War illness (GWI) affects a significant percentage of veterans of the 1991 conflict, but its origin remains unknown. Associated with some cases of GWI are increased incidences of amyotrophic lateral sclerosis and other neurological disorders. Whereas many environmental factors have been linked to GWI, the role of the anthrax vaccine has come under increasing scrutiny. Among the vaccine's potentially toxic components are the adjuvants aluminum hydroxide and squalene. To examine whether these compounds might contribute to neuronal deficits associated with GWI, an animal model for examining the potential neurological impact of aluminum hydroxide, squalene, or aluminum hydroxide combined with squalene was developed. Young, male colony CD-1 mice were injected with the adjuvants at doses equivalent to those given to US military service personnel. All mice were subjected to a battery of motor and cognitive-behavioral tests over a 6-mo period postinjections. Following sacrifice, central nervous system tissues were examined using immunohistochemistry for evidence of inflammation and cell death. Behavioral testing showed motor deficits in the aluminum treatment group that expressed as a progressive decrease in strength measured by the wire-mesh hang test (final deficit at 24 wk; about 50%). Significant cognitive deficits in water-maze learning were observed in the combined aluminum and squalene group (4.3 errors per trial) compared with the controls (0.2 errors per trial) after 20 wk. Apoptotic neurons were identified in aluminum-injected animals that showed significantly increased activated caspase-3 labeling in lumbar spinal cord (255%) and primary motor cortex (192%) compared with the controls. Aluminum-treated groups also showed significant motor neuron loss (35%) and increased numbers of astrocytes (350%) in the lumbar spinal cord. The findings suggest a possible role for the aluminum adjuvant in some neurological features associated with GWI and possibly an additional role for the combination of adjuvants.

Adjuvants, Pharmaceutic↗
PubMed2006

Small interfering RNA targeted to stem-loop II of the 5' untranslated region effectively inhibits expression of six HCV genotypes.

BACKGROUND: The antiviral action of interferon alpha targets the 5' untranslated region (UTR) used by hepatitis C virus (HCV) to translate protein by an internal ribosome entry site (IRES) mechanism. Although this sequence is highly conserved among different clinical strains, approximately half of chronically infected hepatitis C patients do not respond to interferon therapy. Therefore, development of small interfering RNA (siRNA) targeted to the 5'UTR to inhibit IRES mediated translation may represent an alternative approach that could circumvent the problem of interferon resistance. RESULTS: Four different plasmid constructs were prepared for intracellular delivery of siRNAs targeting the stem loop II-III of HCV 5' UTR. The effect of siRNA production on IRES mediated translation was investigated using chimeric clones between the gene for green fluorescence protein (GFP) and IRES sequences of six different HCV genotypes. The siRNA targeted to stem loop II effectively mediated degradation of HCV IRES mRNA and inhibited GFP expression in the case of six different HCV genotypes, where as siRNAs targeted to stem loop III did not. Furthermore, intracytoplasmic expression of siRNA into transfected Huh-7 cells efficiently degraded HCV genomic RNA and inhibited core protein expression from infectious full-length infectious clones HCV 1a and HCV 1b strains. CONCLUSION: These in vitro studies suggest that siRNA targeted to stem-loop II is highly effective inhibiting IRES mediated translation of the major genotypes of HCV. Stem-loop II siRNA may be a good target for developing an intracellular immunization strategy based antiviral therapy to inhibit hepatitis C virus strains that are not inhibited by interferon.

5' Untranslated Regions↗
PubMed2006

Inhibition of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infectivity by peptides analogous to the viral spike protein.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is the cause of an atypical pneumonia that affected Asia, North America and Europe in 2002-2003. The viral spike (S) glycoprotein is responsible for mediating receptor binding and membrane fusion. Recent studies have proposed that the carboxyl terminal portion (S2 subunit) of the S protein is a class I viral fusion protein. The Wimley and White interfacial hydrophobicity scale was used to identify regions within the CoV S2 subunit that may preferentially associate with lipid membranes with the premise that peptides analogous to these regions may function as inhibitors of viral infectivity. Five regions of high interfacial hydrophobicity spanning the length of the S2 subunit of SARS-CoV and murine hepatitis virus (MHV) were identified. Peptides analogous to regions of the N-terminus or the pre-transmembrane domain of the S2 subunit inhibited SARS-CoV plaque formation by 40-70% at concentrations of 15-30 microM. Interestingly, peptides analogous to the SARS-CoV or MHV loop region inhibited viral plaque formation by >80% at similar concentrations. The observed effects were dose-dependent (IC50 values of 2-4 microM) and not a result of peptide-mediated cell cytotoxicity. The antiviral activity of the CoV peptides tested provides an attractive basis for the development of new fusion peptide inhibitors corresponding to regions outside the fusion protein heptad repeat regions.

Amino Acid Sequence↗
PubMed2006

Role of endogenous retroviruses in autoimmune diseases.

Molecular epidemiologic proof that HERVs and other retroelements are involved in autoimmunity or other disorders is complicated by their large numbers in the human genome. As discussed, most HERVs are no longer functional or active because of the accumulation of mutations, frameshifts, and deletions. Detection or quantification of HERV transcripts that may be pathologically involved in a particular autoimmune disease thus is often compromised by the presence in great excess of related, but nonfunctional, RNA. This phenomenon should not deter active work in the field, although it will require development of improved methods to discriminate accurately between closely related RNA transcripts. Development of improved immunologic methods to precisely identify epitopes on autoantigens or rare self-reactive T-cell clones may further implicate HERVs and the other repetitive elements in regulation of the immune system in health and disease.

Animals↗
PubMed2006

Musculoskeletal and autoimmune manifestations of HIV, syphilis and tuberculosis.

PURPOSE OF REVIEW: The HIV pandemic continues to increase at an alarming rate, and is the leading cause of death worldwide from a single pathogen. The number of HIV-1-infected individuals currently exceeds 40 million, the majority of whom live in the developing countries of Asia, sub-Saharan Africa and south America. In the past 5 years, there has concurrently been an increase in the reported cases of tuberculosis and primary and secondary syphilis. This review addresses the musculoskeletal and autoimmune manifestations associated with HIV, syphilis and tuberculosis infections or their treatments. RECENT FINDINGS: During HIV infection the immune system becomes dysfunctional because of the coexistence of immunodeficiency and immune hyperactivity, and a disregulated production or activity of cytokines. Some of these mechanisms explain the development of rheumatic manifestations associated with HIV infection. Highly active antiretroviral therapy changes the course of HIV infection and the spectrum of the HIV-associated rheumatic manifestations. New syndromes such as the immune reconstitution inflammatory syndrome have emerged. HIV, tuberculosis and syphilis infections offer special epidemiological, clinical, and therapeutic challenges. SUMMARY: These observations highlight the complexity and multiplicity of the interactions between the pathogen and host that could result in the development of rheumatic manifestations.

Autoimmune Diseases↗
PubMed2005

Evidence of HIV exposure and transient seroreactivity in archived HIV-negative severe hemophiliac sera.

BACKGROUND: Approximately 25% of hemophiliacs that were frequently exposed to blood clotting factor concentrates (CFCs) contaminated with human immunodeficiency virus (HIV) are presently HIV seronegative. In this study, we sought to determine if some of these individuals were at any time transiently HIV seropositive. In the early to mid-1980s the majority of severe hemophilia patients were exposed to CFCs contaminated with HIV. Although many of these hemophiliacs became HIV-positive, a small percentage did not become infected. To determine if some of these individuals successfully resisted viral infection, we attempted to document the presence of transient HIV reactive antibodies in archived plasma samples (1980-1992) from currently HIV-negative severe hemophiliacs who had a high probability of repeated exposure to HIV contaminated CFC. Archived plasma samples were retrospectively tested using an FDA approved HIV-1Ab HIV-1/HIV-2 (rDNA) enzyme immunoassay (EIA) and a HIV-1 Western blot assay (Wb), neither of which were commercially available until the late 1980s, which was after many of these samples had been drawn. RESULTS: We found that during the high risk years of exposure to HIV contaminated CFC (1980-1987), low levels of plasma antibodies reactive with HIV proteins were detectable in 87% (13/15) of the haemophiliacs tested. None of these individuals are presently positive for HIV proviral DNA as assessed by polymerase chain reaction (PCR). CONCLUSION: Our data suggest that some severe hemophiliacs with heavy exposure to infectious HIV contaminated CFC had only transient low-level humoral immune responses reactive with HIV antigens yet remained HIV-negative and apparently uninfected. Our data supports the possibility of HIV exposure without sustained infection and the existence of HIV-natural resistance in some individuals.

AIDS Serodiagnosis↗
PubMed2005

Safe method for isolation of prion protein and diagnosis of Creutzfeldt-Jakob disease.

Creutzfeldt-Jakob disease (CJD) is a fatal progressive infectious encephalopathy of humans characterized by spongiform degeneration of the brain. Detection of protease-resistant low molecular weight proteins, referred to as 'prions', in the brain is essential for diagnosis. Protease-based methods for prion detection are problematic due to variable susceptibility of prion proteins to proteinase-K digestion. Since CJD brain samples are infectious at all stages of the prion extraction process, we set out to develop a laboratory safe method for prion purification. We lysed the tissues with guanidine thiocyanate followed by phenol extraction of the proteins. Western blotting using prion-specific MAB 3F4 revealed primarily low molecular weight unglycosylated prion (UGP) bands in all CJD cases (19) while the predominant banding in all normal brains (14) represented glycosylated prion (GP). Density readings of the blots revealed the UGP/GP ratio to be significantly different in CJD versus normal brains, with an inverse UGP/GP ratio in CJD. Using this method, we discovered one previously undiagnosed CJD case when we screened 19 brains from the Louisiana State University Alzheimer disease brain bank. Our method is a safe and reliable way of detecting abnormal prion proteins (p<0.0001) and is adaptable to both diagnostic and research laboratories.

Blotting, Western↗
PubMed2005

Peptide inhibitors of dengue virus and West Nile virus infectivity.

Viral fusion proteins mediate cell entry by undergoing a series of conformational changes that result in virion-target cell membrane fusion. Class I viral fusion proteins, such as those encoded by influenza virus and human immunodeficiency virus (HIV), contain two prominent alpha helices. Peptides that mimic portions of these alpha helices inhibit structural rearrangements of the fusion proteins and prevent viral infection. The envelope glycoprotein (E) of flaviviruses, such as West Nile virus (WNV) and dengue virus (DENV), are class II viral fusion proteins comprised predominantly of beta sheets. We used a physio-chemical algorithm, the Wimley-White interfacial hydrophobicity scale (WWIHS) in combination with known structural data to identify potential peptide inhibitors of WNV and DENV infectivity that target the viral E protein. Viral inhibition assays confirm that several of these peptides specifically interfere with target virus entry with 50% inhibitory concentration (IC50) in the 10 microM range. Inhibitory peptides similar in sequence to domains with a significant WWIHS scores, including domain II (IIb), and the stem domain, were detected. DN59, a peptide corresponding to the stem domain of DENV, inhibited infection by DENV (>99% inhibition of plaque formation at a concentrations of <25 microM) and cross-inhibition of WNV fusion/infectivity (>99% inhibition at <25 microM) was also demonstrated with DN59. However, a potent WNV inhibitory peptide, WN83, which corresponds to WNV E domain IIb, did not inhibit infectivity by DENV. Additional results suggest that these inhibitory peptides are noncytotoxic and act in a sequence specific manner. The inhibitory peptides identified here can serve as lead compounds for the development of peptide drugs for flavivirus infection.

Amino Acid Sequence↗
PubMed2005

Synergistic inhibition of human cytomegalovirus replication by interferon-alpha/beta and interferon-gamma.

BACKGROUND: Recent studies have shown that gamma interferon (IFN-gamma) synergizes with the innate IFNs (IFN-alpha and IFN-beta) to inhibit herpes simplex virus type 1 (HSV-1) replication in vitro. To determine whether this phenomenon is shared by other herpesviruses, we investigated the effects of IFNs on human cytomegalovirus (HCMV) replication. RESULTS: We have found that as with HSV-1, IFN-gamma synergizes with the innate IFNs (IFN-alpha/beta) to potently inhibit HCMV replication in vitro. While pre-treatment of human foreskin fibroblasts (HFFs) with IFN-alpha, IFN-beta or IFN-gamma alone inhibited HCMV plaque formation by approximately 30 to 40-fold, treatment with IFN-alpha and IFN-gamma or IFN-beta and IFN-gamma inhibited HCMV plaque formation by 163- and 662-fold, respectively. The generation of isobole plots verified that the observed inhibition of HCMV plaque formation and replication in HFFs by IFN-alpha/beta and IFN-gamma was a synergistic interaction. Additionally, real-time PCR analyses of the HCMV immediate early (IE) genes (IE1 and IE2) revealed that IE mRNA expression was profoundly decreased in cells stimulated with IFN-alpha/beta and IFN-gamma (approximately 5-11-fold) as compared to vehicle-treated cells. Furthermore, decreased IE mRNA expression was accompanied by a decrease in IE protein expression, as demonstrated by western blotting and immunofluorescence. CONCLUSION: These findings suggest that IFN-alpha/beta and IFN-gamma synergistically inhibit HCMV replication through a mechanism that may involve the regulation of IE gene expression. We hypothesize that IFN-gamma produced by activated cells of the adaptive immune response may potentially synergize with endogenous type I IFNs to inhibit HCMV dissemination in vivo.

Antiviral Agents↗
PubMed2005

The aromatic domain of the coronavirus class I viral fusion protein induces membrane permeabilization: putative role during viral entry.

Coronavirus (CoV) entry is mediated by the viral spike (S) glycoprotein, a class I viral fusion protein. During viral and target cell membrane fusion, the heptad repeat (HR) regions of the S2 subunit assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the ectodomain. The formation of this structure appears to drive apposition and subsequent fusion of viral and target cell membranes; however, the exact mechanism is unclear. Here, we characterize an aromatic amino acid rich region within the ectodomain of the S2 subunit that both partitions into lipid membranes and has the capacity to perturb lipid vesicle integrity. Circular dichroism analysis indicated that peptides analogous to the aromatic domains of the severe acute respiratory syndrome (SARS)-CoV, mouse hepatitis virus (MHV) and the human CoV OC43 S2 subunits, did not have a propensity for a defined secondary structure. These peptides strongly partitioned into lipid membranes and induced lipid vesicle permeabilization at peptide/lipid ratios of 1:100 in two independent leakage assays. Thus, partitioning of the peptides into the lipid interface is sufficient to disorganize membrane integrity. Our study of the S2 aromatic domain of three CoVs provides supportive evidence for a functional role of this region. We propose that, when aligned with the fusion peptide and transmembrane domains during membrane apposition, the aromatic domain of the CoV S protein functions to perturb the target cell membrane and provides a continuous track of hydrophobic surface, resulting in lipid-membrane fusion and subsequent viral nucleocapsid entry.

Amino Acid Sequence↗
PubMed2005

Seroreactivity to A-type retrovirus proteins in a subset of cats with hyperthyroidism.

The thyroid gland is afflicted in several endocrine, autoimmune, and malignant diseases. Previous studies detected immunoreactivity against proteins of a human intracisternal A-type retroviral particle type-I (HIAP-I) in serum samples from the majority of patients with Graves' disease, an autoimmune disease of the thyroid that can also affect other organs, most prominently the eyes. To determine whether hyperthyroid animals might provide a model for the retroviral involvement in thyroid autoimmunity, serum samples from 32 cats (21 hyperthyroid and 11 controls) and 10 hypothyroid dogs were examined for immunoreactivity with HIAP-I using a Western blot technique. Of the 21 hyperthyroid cats 15 (71.4%) were HIAP-I positive, while only 2 of 11 (11.8%) control animals without endocrine pathology were positive. No significant correlations were seen between HIAP seroreactivity and serum thyroid hormone levels (T3 and T4), age, gender, treatment history, vaccination status, or weight. No seroreactivity to HIAP-I was detected in hypothyroid dogs. An examination of HIAP-I reactivity in feline leukemia virus (FeLV)-seroconverting cats found that 7/9 (78%) animals viremic for FeLV-A showed an alteration in HIAP serology, whereas only 1/7 (14%) nonviremic animals showed a change in HIAP-I serology. These results suggest that it may be possible to develop an animal (feline) model for the role of retroviruses in thyroid autoimmune diseases.

Animals↗
PubMed2005

Dengue in the Dominican Republic: epidemiology for 2004.

The Dominican Republic (DR) has experienced tremendous increase in the number of cases of dengue virus (DENV) reported in the past few years. There are four serotypes of DENV (1-4), and each can cause classic dengue fever (DF), dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS). DENV-1-4 are currently circulating in the DR; however, the Department of Epidemiology of the Dominican Republic (DIEGPI) has been able to isolate only DENV-2 and DENV-4. In the DR, the development of DENV infection occurred primarily in the second semester of the year. Since the DR instituted a vigilant approach to dengue infections, there have been three major outbreaks detected: one in 1998, one in 2000, and one in 2002. DF is the clinical presentation most currently seen at clinics, accounting for about 75% of cases, while patients with DHF account for about 19% of cases seen. Currently, there are seven provinces in which the total number of dengue cases per 100,000 inhabitants is higher than 32. With a vigilant approach, the DR should continue to see success in controlling the DENV outbreak.

DNA, Viral↗
PubMed2005

Of mice, cats, and men: is human breast cancer a zoonosis?

Mouse mammary tumor virus (MMTV), a member of the betaretroviridae, is the most common cause of breast cancer (BC) in mice. MMTV is transmitted in mice both in the germline as endogenous proviruses and exogenously as infectious virions. Here, we review a variety of evidence accumulated for six decades that has suggested that a human homologue of MMTV may exist. The findings include recent studies from several independent laboratories that have detected sequences very closely related to MMTV in DNA isolated from human BC tumors. Other laboratories, however, have failed to detect the MMTV-related sequences in human DNA samples, and conclusive evidence for a human mammary tumor virus has been elusive. We also reviewed additional studies, suggesting that betaretroviruses are present in a much wider range of species than previously known, including rodents, felines, and primates. The observation that a subset of cats may be infected with a close homologue of MMTV may be of epidemiological significance for human BC. Cats may become infected by MMTV from mice, and in turn may transmit the virus to humans, possibly after selection for variants with an expanded host range.

Animals↗
PubMed2005

Human, rhesus macaque, and feline sequences highly similar to mouse mammary tumor virus sequences.

Sequences highly similar (>95%) to the mouse mammary tumor virus (MMTV) env gene have been amplified from human DNA samples, including DNA samples from patients with breast cancer (BC) and persons who did not have BC. The sequences from human DNA were distinct from the MMTV sequences used as controls in these PCR reactions, indicating that these results are not simply due to contamination. In addition to both, mouse and human-related sequences were also amplified from some monkey and cat genomic DNA samples. These products were shown to be distinct from, but highly related to, the MMTV env gene, whereas, testing of other sources (lambda phage, snake, cockroach, sea urchin, chicken, or dog) demonstrated no specific amplification. A sequence 90% similar to the MMTV group antigen gene (gag) was amplified from cat DNA. These results indicate that DNA from vertebrate species other than rodents, including some but not all humans, monkeys, and cats, can contain sequences closely related to MMTV.

Animals↗
PubMed2005

Involvement of human intracisternal A-type retroviral particles in autoimmunity.

Prior studies have linked retroviruses to various arthropathies and autoimmune diseases. Sjögren's syndrome (SS), a systemic autoimmune disease, is characterized by aggressive infiltration of lymphocytes into the salivary and lacrimal glands, resulting in destruction of the glands and dry mouth and eyes (sicca syndrome). The infiltrating lymphocytes in SS may become overtly malignant, and thus, the incidence of lymphoma is greatly increased in SS patients. A human intracisternal A-type retroviral particle type I (HIAP-I) has been isolated from persons with SS. HIAP-I shares a limited number of antigenic epitopes with human immunodeficiency virus (HIV), but is distinguishable from HIV by morphological, physical, and biochemical criteria. A substantial majority of patients with SS or systemic lupus erythematosus (SLE) have serum antibodies to the proteins of this human retrovirus. Fewer than 3% of the normal blood donor population have antibodies to any HIAP-associated proteins. A second type of a human intracisternal A-type retrovirus, HIAP-II, was detected in a subset of patients with idiopathic CD4 lymphocytopenia (ICL), an AIDS-like immunodeficiency disease. Most HIAP-II positive ICL patients were also antinuclear antibody positive. Reviewed here are additional studies from several laboratories suggesting that HIAP or related viruses may be involved in SLE and other autoimmune conditions. Additionally, results of comprehensive surveys of autoimmune patients to determine seroreactivity to HIAP, and other human retroviruses, including HIV and human T-lymphotropic virus type I, are reported.

Autoimmune Diseases↗
PubMed2005

Alpha interferon inhibits translation mediated by the internal ribosome entry site of six different hepatitis C virus genotypes.

Certain genotypes of hepatitis C virus (HCV) respond less often than others to treatment with interferon (IFN). The mechanisms for this differential response are not known. In this report antiviral effects of IFN-alpha2b on translation were examined in a hepatic cell line using chimeric clones of internal ribosome entry site (IRES) sequences from six different HCV genotypes and the green fluorescence protein (GFP) gene. As a control, IFN action at the level of the IRES was examined in the presence of different cytokines. It was determined that IFN-alpha2b specifically inhibited the translation of GFP mediated by IRES sequences from six major HCV genotypes in a concentration-dependent manner. Other cytokines including tumour necrosis factor alpha, transforming growth factor beta 1, interleukin 1 and interleukin 6 have no inhibitory effect. The inhibition of translation in these experiments was not due to extensive intracellular degradation of IRES-GFP mRNA. These results suggest that the antiviral action of IFN-alpha2b blocks IRES-mediated translation and this effect is the same among HCVs of other genotypes.

Genotype↗
PubMed2005

Identification and characterization of the putative fusion peptide of the severe acute respiratory syndrome-associated coronavirus spike protein.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a newly identified member of the family Coronaviridae and poses a serious public health threat. Recent studies indicated that the SARS-CoV viral spike glycoprotein is a class I viral fusion protein. A fusion peptide present at the N-terminal region of class I viral fusion proteins is believed to initiate viral and cell membrane interactions and subsequent fusion. Although the SARS-CoV fusion protein heptad repeats have been well characterized, the fusion peptide has yet to be identified. Based on the conserved features of known viral fusion peptides and using Wimley and White interfacial hydrophobicity plots, we have identified two putative fusion peptides (SARS(WW-I) and SARS(WW-II)) at the N terminus of the SARS-CoV S2 subunit. Both peptides are hydrophobic and rich in alanine, glycine, and/or phenylalanine residues and contain a canonical fusion tripeptide along with a central proline residue. Only the SARS(WW-I) peptide strongly partitioned into the membranes of large unilamellar vesicles (LUV), adopting a beta-sheet structure. Likewise, only SARS(WW-I) induced the fusion of LUV and caused membrane leakage of vesicle contents at peptide/lipid ratios of 1:50 and 1:100, respectively. The activity of this synthetic peptide appeared to be dependent on its amino acid (aa) sequence, as scrambling the peptide rendered it unable to partition into LUV, assume a defined secondary structure, or induce both fusion and leakage of LUV. Based on the activity of SARS(WW-I), we propose that the hydrophobic stretch of 19 aa corresponding to residues 770 to 788 is a fusion peptide of the SARS-CoV S2 subunit.

Amino Acid Sequence↗