Environmental factors in the development of autism spectrum disorders.

Autism spectrum disorders (ASD) are highly heterogeneous developmental conditions characterized by deficits in social interaction, verbal and nonverbal communication, and obsessive/stereotyped patterns of behavior and repetitive movements. Social interaction impairments are the most characteristic deficits in ASD. There is also evidence of impoverished language and empathy, a profound inability to use standard nonverbal behaviors (eye contact, affective expression) to regulate social interactions with others, difficulties in showing empathy, failure to share enjoyment, interests and achievements with others, and a lack of social and emotional reciprocity. In developed countries, it is now reported that 1%-1.5% of children have ASD, and in the US 2015 CDC reports that approximately one in 45 children suffer from ASD. Despite the intense research focus on ASD in the last decade, the underlying etiology remains unknown. Genetic research involving twins and family studies strongly supports a significant contribution of environmental factors in addition to genetic factors in ASD etiology. A comprehensive literature search has implicated several environmental factors associated with the development of ASD. These include pesticides, phthalates, polychlorinated biphenyls, solvents, air pollutants, fragrances, glyphosate and heavy metals, especially aluminum used in vaccines as adjuvant. Importantly, the majority of these toxicants are some of the most common ingredients in cosmetics and herbicides to which almost all of us are regularly exposed to in the form of fragrances, face makeup, cologne, air fresheners, food flavors, detergents, insecticides and herbicides. In this review we describe various scientific data to show the role of environmental factors in ASD.

Detection of anthrax toxin genetic sequences by the solid phase oligo-probes.

PURPOSE: There is an urgent need to detect a rapid field-based test to detect anthrax. We have developed a rapid, highly sensitive DNA-based method to detect the anthrax toxin lethal factor gene located in pXO1, which is necessary for the pathogenicity of Bacillus anthracis. MATERIALS AND METHODS: We have adopted the enzyme-linked immunosorbent assay (ELISA) so that instead of capturing antibodies we capture the DNA of the target sequence by a rapid oligo-based hybridization and then detect the captured DNA with another oligoprobe that binds to a different motif of the captured DNA sequences at a dissimilar location. We chose anthrax lethal factor endopeptidase sequences located in pXO1 and used complementary oligoprobe, conjugated with biotin, to detect the captured anthrax specific sequence by the streptavidin-peroxidase-based colorimetric assay. RESULT: Our system can detect picomoles (pMoles) of anthrax (approximately 33 spores of anthrax) and is >1000 times more sensitive than the current ELISA, which has a detection range of 0.1 to 1.0 ng/mL. False positive results can be minimized when various parameters and the colour development steps are optimized. CONCLUSION: Our results suggest that this assay can be adapted for the rapid detection of minuscule amounts of the anthrax spores that are aerosolized in the case of a bioterrorism attack. This detection system does not require polymerase chain reaction (PCR) step and can be more specific than the antibody method. This method can also detect genetically engineered anthrax. Since, the antibody method is so specific to the protein epitope that bioengineered versions of anthrax may not be detected.

Seroprevalence of hepatitis B and C genotypes among young apparently healthy females of karachi-pakistan.

INTRODUCTION: Although the prevalence of hepatitis virus infections in Pakistan is still unknown, limited data indicate that the exposure rate to HBV is 35-38% with 4% being carriers and 32% having anti-HBV surface antibodies through natural conversion [1, 2, 3]. Studies in Pakistan have shown that the prevalence rate of HCV is 4.8-14% for, and that it is continuously increasing. Hence there is an urgent need to create awareness about the prevalence of both hepatitis B and C, and to develop preventive measures aimed at minimizing the prevalence of these diseases in the country. STUDY DESIGN: Prospective, descriptive study. The study took place from March 2002 till October 2006 at two university campuses in Karachi. MATERIALS AND METHODS: A total of 4000 healthy female students were screened for HBs Ag, anti-HBs antibodies and anti-HCV antibodies by rapid immunochromatography, ELISA and PCR. RESULTS: A total of 3820 volunteers (95.5%) were negative by all three methods, 181 (4.5%) tested positive for HB surface antigen and 20 (0.5%) were positive for anti HB surface antibodies; 208 volunteers (5.2%) were positive for HCV. Double infection with HBV and HCV was found in only one patient (0.025%). Out of 180 HBs antigen positive samples 151 (83.89%) were genotype D, 28 (15.56%) showed mixed infection with genotypes B and D, and one patient (0.56%) showed mixed infection with genotypes C and D. Out of 208 samples positive for HCV antibodies, 107 (51.44%) were genotype 3a, 50 (24.04%) were mix of genotype 3a and 3b, 33 (15.87%) were genotype 3b, 10 (4.81%) were genotype 1b while, 8 (3.84%) samples could not be typed. CONCLUSION: Although the presence of these pathogenic viruses was not very high in our young healthy female population, it is still a matter of concern to control the unregulated spread of these deadly infections by promoting increased awareness and regular immunization programs in the community. Local manufacturing of vaccines and related products may reduce these infections.

In situ polymerase chain reaction and hybridization to detect low-abundance nucleic acid targets.

This unit presents a novel approach for detecting low-abundance nucleic acid targets in nuclear and cytoplasmic regions by amplification of specific target sequences using an in situ polymerase chain reaction (ISPCR). If the target sequence is RNA, ISPCR is preceded by in situ reverse transcription. Following ISPCR, in situ hybridization is performed. An describes a variant in situ method for simultaneously reverse transcribing and amplifying RNA transcripts using recombinant Thermos thermophilos (rTth) polymerase. In situ hybridization and detection of amplified targets is also described.

Multicenter comparison of PCR assays for detection of human herpesvirus 8 DNA in semen.

Reported prevalences of human herpesvirus 8 (HHV-8) (Kaposi's sarcoma-associated herpesvirus) in semen have ranged widely. This is possibly due to differences in assay sensitivity, geographic or population-based differences in the true presence of the virus in semen, and PCR contamination. This study assessed interlaboratory sensitivity and reproducibility in the analysis of blinded experimental panels, each consisting of 48 specimens and being composed of semen specimens from different healthy artificial-insemination donors (n = 30) and human immunodeficiency virus (HIV)-infected patients (n = 7) plus positive (n = 4) and negative (n = 7) controls. The experimental panels analyzed in each laboratory were identical except for being independently coded. Of 10 experiments done in five laboratories, 5 experiments from three laboratories had evidence of PCR contamination; all instances of contamination were in the context of nested PCR procedures. In the experiments with no false-positive results, HHV-8 DNA was detected in three (8%) of the 37 semen specimens (two from artificial-insemination donors and one from an HIV-positive patient) but in only 3 (1.6%) of the 184 PCRs in which these specimens were analyzed. This suggests that HHV-8 DNA is present in semen at concentrations that can be too low to allow its consistent detection. This study emphasizes the importance of performing blinded, multi-institution experiments to provide a coherent basis for comparing results and to motivate standardization of methods.

Detection of nucleic acids in cells and tissues by in situ polymerase chain reaction.

The solution-based polymerase chain reaction (PCR) method for amplification of defined gene sequences has proved a valuable tool not only for basic researchers but also for clinical scientists. Using even a minute amount of DNA or RNA and choosing a thermostable enzyme from a large variety of sources, one can enlarge the amount of the gene of interest, which can be analyzed and sequenced. Therefore, genes, or segments of gene sequences present only in a small sample of cells or small fraction of mixed cellular populations can be examined. One of the major drawbacks of the solution-based PCR technique is that the procedure does not allow for the association of amplified signals of a specific gene segment with the histological cell type(s) (1-2). For example, it would be advantageous to determine what types of cells in the peripheral blood circulation or in pathological specimens carry HIV-1 gene sequences, a vector used for gene therapy, an aberrant gene in a leukemia patient, or to determine the percentage of leukemia cells present following antitumor therapy.

Viral burden and disease progression in HIV-1-infected patients with sickle cell anemia.

The spleen and lymph nodes are major sites of human immunodeficiency virus type 1 (HIV-1) replication, mutation, and genetic variation in vivo. If a major portion of the lymphatic tissue, such as the spleen, is removed or otherwise is unavailable for invasion by the HIV-1 virus, will the course of the infection be altered, resulting in a prolonged symptom-free interval or even increased survival? The spleen of most adults with sickle cell anemia (SS) is nonfunctional due to recurrent episodes of microinfarction. If autosplenectomized SS patients are exposed to HIV-1, they may be ideal candidates to examine the question of whether absence of splenic function at the time of infection will positively alter the course of HIV-1-related disease. All SS patients with a diagnosis of HIV-1 infection at five university sickle cell centers were included in the patient cohort. Patients in active treatment or in follow-up (group A, n = 11) underwent a series of quantitative viral studies to determine their HIV-1 viral burden. The studies included the branched-DNA signal amplification assay, quantitative DNA-polymerase chain reaction (PCR), quantitative reverse transcription (RT)-initiated-PCR, and in situ PCR. All patients who died of the complications of the acquired immunodeficiency syndrome (AIDS) or of SS, lost to follow-up, or were otherwise unavailable for study (Group B: n = 7) were included in the total patient group. None of the patients in group B underwent quantitative viral studies. In addition, a control population (group C, n = 36) of HIV-1-infected African Americans without SS, of similar age and gender to the SS patients, were compared with the study population for outcomes. In eight of 11 active patients (group A), the CD4+ T-lymphocyte counts were normal and viral burdens were low for an average of 10.25 years following diagnosis. These eight patients all from group A were the only long-term nonprogressors (44%) among a total of 18 SS patients (groups A and B). In group C (control), only five patients of 36 were long-term nonprogressors (13.9%). Five patients (28%) of the total SS group (groups A and B) succumbed to AIDS. One of the five was from Group A. The evaluation of a limited number of adult individuals suggests that a significant proportion of HIV-1-seropositive SS patients (44%) may be asymptomatic long-term nonprogressors. In these patients, the CD4+ T-lymphocyte counts remained high and their viral burdens were remarkably lower than in non-SS HIV-1-seropositive individuals. Whereas this study does not prove an "autosplenectomy" hypothesis, it suggests that in patients with both SS and HIV-1 infection, the retroviral disease may be ameliorated by host factors of which absence of splenic function prior to HIV-1 infection may be one.

Molecular cloning of platelet factor XI, an alternative splicing product of the plasma factor XI gene.

Platelet factor XI is associated with the platelet plasma membrane and has an apparent Mr (220,000 nonreduced, 55,000 reduced) different from that of plasma factor XI. However, the site of synthesis and the nature of platelet factor XI are not known. Using reverse transcriptase polymerase chain reaction, 12 out of 13 exons (all except exon V) coding for mature plasma factor XI were amplified from human platelet mRNA. The sequence of each of these exons was identical to that of plasma factor XI. In situ amplification and hybridization of factor XI mRNA was positive for exon III and negative for exon V in platelets and negative for both exons in other blood cells. By Northern hybridization, a factor XI mRNA transcript of approximately 1.9 kilobases was detected in megakaryocytic cells, and one of approximately 2.1 kilobases was detected in liver cells. Factor XI cDNA was cloned from a megakaryocyte library and sequenced. Exon V was absent, and the splicing of exon IV to exon VI maintained the open reading frame without alteration of the amino acid sequence except for the deletion of amino acids Ala91-Arg144 within the amino-terminal portion of the Apple 2 domain. Thus, platelet factor XI is an alternative splicing product of the factor XI gene, localized to platelets and megakaryocytes but absent from other blood cells.

Localization of human herpesvirus type 8 (HHV-8) in the Kaposi's sarcoma tissues and the semen specimens of HIV-1 infected and uninfected individuals by utilizing in situ polymerase chain reaction.

Kaposi's sarcoma (KS) is a form of skin cancer, most commonly found in individuals suffering from acquired immunodeficiency syndrome, or AIDS. However, before the worldwide infection of human immunodeficiency virus (HIV), the rare occurrence of KS was confined to two distinct groups of individuals. In the Western world, the classical form of KS was often found in older men (60-70 years of age) from the Mediterranean area. Another form called endemic KS, was found in Equatorial Africa. Currently, the most common cases of KS are found in individuals suffering from AIDS. This is called AIDS-associated KS. Between 30 and 40% of male, homosexual AIDS patients suffer from AIDS-associated KS. KS is also occasionally diagnosed in transplant patients receiving immunosuppressive drugs (to keep their body from rejecting the foreign organ). As opposed to cases of classic and endemic KS, the KS in AIDS patients progresses very quickly, often with a fatal outcome. Human herpesvirus type 8 (HHV-8) has been implicated as the cause of Kaposi's sarcoma (KS), but the exact connection of the virus to the neoplasm is not known. The virus has been detected within the sarcoma skin lesions, but has additionally been seen in peripheral blood cells, semen samples, prostate tissue, and other types of soft tissue tumors. In this study, we evaluated HHV-8 within the skin lesion of KS as well as in semen specimens obtained from HIV-1 infected and uninfected specimens from HIV-1-seronegative individuals. Twenty-eight tissue samples representing AIDS-associated, endemic KS, and six non-KS patients were collected for observation from different centers throughout the world. The tissues were examined utilizing in situ polymerase chain reaction (ISPCR) and hybridization to identify and localize the herpesvirus within the KS lesions. With the use of the sensitive ISPCR technique, HHV-8 DNA was detected in the spindle cells within the nodular skin lesions, as well as in the microvascular endothelial cells which line small vessels within the lesions in all forms of KS. In addition, we analysed semen specimens from HIV-1 infected and uninfected men, our analyses revealed that HHV-8 was present in the significant proportions of the HIV-1-infected-individuals' sperm, as well as in the mononuclear cells of the semen specimens. HHV-8 DNA was demonstrated, by ISPCR, in KS lesions as well as in seminal mononuclear cells and sperm of significantly high proportion of HIV-1-infected men. What role the presence of HHV-8 in the sperm cells plays in the sexual transmission of this herpesvirus will require further study. However, the reports which demonstrate that KS lesions can develop in infants of only a few weeks of age, increases the possibility that this agent may be vertically transmitted. It can be suggested that HHV-8 is relatively ubiquitous and its frequency increases with the increasing immunosuppression.

Self-seal reagent: evaporation control for molecular histology procedures without chambers, clips or fingernail polish.

Sensitive nucleic acid based detection methods such as in situ PCR, in situ RT-PCR and PRINS have great potential in the areas of developmental biology, pathogenesis and diagnostics. However, control of evaporation from in situ reactions is critical to ensure reliable data. Self-Seal Reagent, a component added directly to the in situ reaction mixture, effectively controls evaporation during in situ procedures by creating an evaporation-limiting barrier around the periphery of a standard cover glass as the reaction proceeds. At the end of the procedure, the cover glass is easily removed by soaking in an aqueous solution. A model is presented for how Self-Seal Reagent controls evaporation while maintaining reagent concentrations. Self-Seal Reagent is shown to be effective in the detection of HIV sequences in cells by in situ PCR.

Natural immunity against human immunodeficiency viruses: prospects for AIDS vaccines.

We have hypothesized and have presented evidence that there may be another form of immunity, other than humoral and cellular immunities, which operates against retroviruses. In order to distinguish it from the traditional immune responses, we have named this form of immunity "molecular immunity". The major goal of this hypothesis is to better define the "messenger molecules" that are critical in forming the molecular immunity against retroviruses, and to further determine the activation pathways of this relatively unexplored form of immunity. We have provided evidence that this natural immunity against retroviruses and specifically against HIV-1, can be activated and optimized, and have made some interesting observations. We believe that resistance to HIV-1 and to other retroviruses can be induced by various means, including low dose exposure, infection with replication defective viruses and exposure to non-pathogenic but genetically related viruses.

Detection of HIV in oral mucosal cells.

OBJECTIVE: To determine the prevalence of HIV DNA and RNA and the morphologic localization of HIV in the oral cavity of HIV-seropositive subjects. DESIGN: A cross-sectional analysis of saliva, buccal scrapings and buccal biopsies from HIV-seropositive injecting drug users (IDUs). SUBJECTS AND METHODS: Whole saliva, buccal mucosal scrapings and buccal biopsies were obtained from HIV-seropositive and seronegative IDUs. Presence of HIV DNA and RNA was assessed by polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR). RT in situ PCR was used to detect HIV tat/rev RNA in buccal mucosal scrapings. Host-cell integrated HIV-proviral DNA in buccal biopsies was detected by in situ PCR. Presence of intact HIV viral particles in buccal scrapings was assessed by electron microscopy. RESULTS: HIV DNA was detected in 40% (18/45) and HIV RNA in 69.2% (25/36) of saliva samples from HIV-seropositive IDUs. Viral particles consistent with HIV were localized in inter-epithelial spaces by electron microscopy. RT in situ PCR revealed the presence of HIV tat/rev RNA in 36% (8/22) of the seropositive samples tested. CONCLUSIONS: Our results suggest that epithelial cells can be productively infected by HIV. Epithelial cells in buccal mucosa may acquire HIV in the basal layers through contact with submucosal HIV-positive lymphocytes and/or Langerhans' cells. HIV infection may also spread by inter-epithelial cell contact. As HIV infected cells mature they travel to more superficial layers and are shed into the oral cavity.

Absence of the inducible form of nitric oxide synthase in the brains of patients with the acquired immunodeficiency syndrome.

A majority of human immunodeficiency virus type I (HIV-1)-infected-individuals manifest a plethora of central nervous system (CNS) diseases unrelated to opportunistic infections, including acquired immune deficiency syndrome (AIDS)-dementia complex (ADC), encephalitis, and various other disorders of the CNS. A series of devastating clinical conditions in the CNS of certain HIV-1-infected-individuals may be caused by infection of cells in the brain parenchyma. ADC is characterized by cognitive dysfunction, motor difficulties, coordination abnormalities and other neurological signs and symptoms, which develop in many HIV-1-infected-individuals. The precise molecular mechanisms leading to AIDS dementia remain incompletely explained. Various mechanisms including cytokine dysregulation, toxic effects of viral proteins and release of certain toxic substances from macrophages, especially nitric oxide, have been implicated as pathogenic mediators in the development of ADC. We have examined post mortem CNS tissues collected from 22 patients, previously diagnosed with AIDS, to explore if nitric oxide is responsible for the observed pathology in ADC. As controls, we utilized tissues collected from the brains of patients who expired without AIDS or other CNS pathologies. In addition, we also utilized post-mortem brain tissues from eight patients who were diagnosed with multiple sclerosis (MS) and were found to express inducible nitric oxide synthase (iNOS) in our previous studies, as positive controls. Highly sensitive in situ reverse transcriptase-initiated polymerase chain reaction (RT-IS-PCR) studies demonstrated that iNOS mRNA was present in the CNS tissues from all the positive MS controls, but were absent in all 22 specimens from AIDS patients, as well as in the brain tissues from normal controls. We have also analyzed the tissues for the presence of the NO reaction product, nitrotyrosine, to evaluate the presence of a protein nitrosalation adduct. Nitrotyrosine was not demonstrable in any of the AIDS brains. These findings indicate that iNOS may not play a significant role in the neuropathogenesis of most cases of ADC.

Prevention of experimental allergic encephalomyelitis by targeting nitric oxide and peroxynitrite: implications for the treatment of multiple sclerosis.

In this study we provide further evidence associating activated cells of the monocyte lineage with the lesions of multiple sclerosis (MS). Using a combination of immunohistochemistry and reverse transcriptase-dependent in situ polymerase chain reaction analysis, we have identified monocytes expressing inducible nitric oxide synthase (iNOS) to be prevalent in the plaque areas of post mortem brain tissue from patients with MS. In addition, we have obtained evidence of the nitration of tyrosine residues in brain areas local to accumulations of iNOS-positive cells. In parallel studies we have assessed the effects of inhibitors of iNOS induction, as well as scavengers of nitric oxide and peroxynitrite in the experimental allergic encephalomyelitis model. Significant therapeutic effects were seen with the inhibitor of iNOS induction, tricyclodecan-9-xyl-xanthogenate, a nitric oxide scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and a peroxynitrite scavenger, uric acid. In particular, treatment with high doses of uric acid virtually prevented clinical symptoms of the disease. Together with our demonstration of the presence of activated macrophages expressing high levels of iNOS and evidence of peroxynitrite formation in brain tissue from patients with MS, these findings are of importance in the development of approaches to treat this disease.