Techniques Including Polymerase Chain Reaction Research Articles

Overview of Mycotic Keratitis.

Keratomycosis is a serious corneal infection associated with high ocular morbidity that can lead to severe vision loss. It is estimated to affect more than 1 million patients annually, most commonly occurring in tropical climates, and represents a growing threat to patients worldwide. Despite aggressive medical management, fungal infections have a higher rate of perforation requiring surgical intervention compared with other infectious etiologies. Early diagnosis and appropriate treatment are keys to preserving vision and saving patients' eyes.Timely diagnosis of fungal keratitis helps minimize corneal damage and scarring and increases the likelihood of a favorable outcome. Studies have shown that correct identification of fungal infections is often delayed up to 2 to 3 weeks after initial presentation. This leads to incorrect or ineffective treatment for many patients. Diagnostic techniques explored in this study include corneal scrapings with staining and culture, visualization with in vivo confocal microscopy, molecular diagnostic techniques including polymerase chain reaction, and recently developed omics-based technologies.Treatment of fungal keratitis begins with topical antifungals. Medical management has been proven to be effective, but with limitations including poor drug penetration and low bioavailability. Cases that do not respond to topical therapy require more invasive and novel treatments to control the infection. We review the clinical trials that have shaped current practice patterns, with focus on the efficacy of topical natamycin as the primary therapy for filamentous fungal keratitis. We explore additional management strategies such as localized intrastromal and intracameral injections of antifungal medications, photodynamic therapy, and surgical intervention.

Abstract 423: Cell state-specific genomics to define plasticity along the epithelial-mesenchymal transitioncontinuum

Abstract The epithelial-to-mesenchymal transition (EMT) is a biological process that allows epithelial cells to convert into a mesenchymal phenotype, acquiring increased mobility and the capacity to migrate. EMT has historically been represented as binary states; however, a growing consensus supports an EMT continuum involving multiple intermediate stable states. While we lack a comprehensive understanding of the EMT continuum, several reports indicate that the late stable states lead to optimal stem cell traits for cancer. In addition, it has been suggested that EMT heterogeneity within tumor cell populations is associated with poor patient prognosis. Hence, there is a clear need to define the genetic drivers of EM plasticity in heterogeneous cell populations along the EMT continuum. To fulfill this need, we will collect and analyze single cell RNA sequencing (scRNA-seq) data from EMT-cell-state specific genetic CRISPR screens on EMT-heterogeneous cell populations. Here, we apply a series of cloning techniques including polymerase chain reaction and Gibson ligation to engineer an adeno-associated virus (AAV) plasmid containing the avian TVA receptor gene under control of the N-cadherin promoter. Since N-cadherin is predominantly expressed in mesenchymal cells, this AAV enables us to generate cell lines that will exclusively express the TVA receptor when in a mesenchymal state. Consequently, we will be able to deliver mesenchymal-specific CRISPR gene edits via an avian retrovirus-derived RCAS system. Citation Format: Destinee K. Williams, Nicholas Hou. Cell state-specific genomics to define plasticity along the epithelial-mesenchymal transitioncontinuum [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 423.

Role of Human Papillomavirus in Carcinogenesis

Since a decade ago, it has been observed that the incidence of cancers caused by human papillomavirus (HPV) has seen an uptick, making it a point of concern from the public health perspective. Numerous cancers, including oral and cervical cancers, have been linked to HPV. HPV is carcinogenic due to its ability to induce abnormal expression of several genes, including nuclear factor-kappa B, nuclear factor erythroid 2-related factor 2, p16, p53, RB1 and some microRNA genes. Early cancer treatment involves identifying the up- or downregulation of various factors using techniques including polymerase chain reaction (PCR), reverse transcriptase PCR, p16 immunostaining and in situ hybridisation. The virus achieves its breakthrough by taking advantage of various types of cellular enzymes, enzymatic activities and their association or disassociation with other factors in order to up/downregulate enzyme levels. The methods used for screening and detection still need to have high sensitivity and high accuracy because the amount of factors showing change is remarkably minute. Vaccines can remarkably reduce the risk of HPV infection when administered as directed and in the recommended number of doses.

A Comparative Microbiological Quality Assessment of Frozen and Fresh Fishes from Erbil City

Background: Since ancient times, the majority of humans have been categorized as pescatarians, depending mostly on seafood and fish as well as their byproducts. Fish is currently in greater demand as it is a good source of protein, which is necessary for maintaining the health of muscles, organs, and blood vessels. Due to their sensitive tissues and aquatic environment, fish are especially prone to microbial infections. It has been suggested that one way to assess the quality of fish is through the monitoring of these bacteria. Objectives: The current study aimed to examine the microbiological quality of fresh and frozen fishes. Materials and Methods: A comparative study was conducted to evaluate the microbial quality of fresh and frozen common carps to raise food safety concerns, Iraq-Erbil city. A total of 50 samples of fishes were collected from local markets and breeding ponds from September 2021 to January 2022. The most probable number (MPN) method was used for the estimation of the total coliform, whereas the horizontal method was used for the enumeration of Salmonella spp. Results: In this study, 53% of frozen samples contaminated with the coliform, whereas 15% of the fresh common carps were contaminated with coliform and passed the acceptable range (<100 MPN/g). Salmonella spp. was not found in any fresh samples, although it was recorded in 20% of the frozen common carps. Conclusion: Further investigations are required with molecular techniques including polymerase chain reaction to determine the species and type of contamination in frozen fishes.

Prevalence of bacterial infections and the use of multiplex PCR assay for rapid detection of pathogens in cultured fish in Ghana.

The modern and rapid avenue for detecting pathogens provided by molecular genetic techniques including polymerase chain reaction (PCR) was explored in the present study to identify prevalent disease pathogens, from six aquaculture farms and in two commonly cultured fish in Ghana. The specific detection was carried out directly on clinical samples of naturally infected fish (O. niloticus and C. gariepinus) based on syber-mix reaction protocol in traditional PCR. Molecular diagnostic techniques allowed the detection of the six most common and important bacterial pathogens in aquaculture farms in Ghana. Also, three of the pathogens (Streptococcus agalactiae, Streptococcus iniae, and Staphylococcus aureus) were simultaneously isolated in a multiplex reaction. The results indicated 90-100% sensitivity and specificity for each of the six bacterial pathogens tested. Streptococcosis and motile aeromonad septicemia were found to be highly prevalent in most aquaculture farms in Ghana with severity in infections traced to the 85.7% and 14.9% co-infections with all six target pathogens in catfish and tilapia respectively. The prevalence rate of infections significantly correlated with variations in salinity, conductivity, and dissolved oxygen concentrations in the thermal stressed condition of the culture water. Multiplex techniques employed in this study represent one of the first to be used by a fish health laboratory in Ghana for rapid detection of pathogens in diseased fish and could be a useful alternative to the culture-based method for routine diagnosis of fish diseases in Ghana.

Prevalence of Microsporum canis from Pet Cats in Small Animal Hospitals, Chiang Mai, Thailand.

Dermatophytosis is a disease caused by dermatophytes, a group of fungi that can cause disease both in humans and animals. The important genera that are pathogenic in animals include Trichophyton and Microsporum. Microsporum canis is an important species because it can cause zoonosis and is commonly found in domestic animals. Cats, which live very close to humans, may expose humans to this pathogen. This research focused on the epidemiology of M. canis found in cats. Hair samples were collected via the Mackenzie technique from cats with and without skin lesions, preliminarily examined with 10% KOH preparation, and cultured for fungal identification. Samples were confirmed with molecular techniques including polymerase chain reaction, gel electrophoresis, and sequencing. Samples were collected from 138 cats located in 93 households, 43 from cats with skin lesions (31.16%) and 95 from cats without skin lesions (68.84%). Eighteen cats with lesions (13.04%) and ten cats without lesions (7.2%) were found to carry M. canis. In eleven of the eighteen cats both with skin lesions and positive for M. canis (61.11%), the pathogen was found both at the site of the lesion and at other sites in the body. Because the pathogen can be found in the hair of cats with and without skin lesions, owners, keepers, veterinarians, and others who come into contact with these animals are at risk of infection if they are not aware or do not take precautions after contact with them.

Evaluation of a Salmonella Strain Isolated from Honeybee Gut as a Potential Live Oral Vaccine Against Lethal Infection of Salmonella Typhimurium.

In this research, Salmonella species were isolated from the animal, insect and human enteric sources in Faisalabad, Punjab, Pakistan. These species were characterized by different microbiological and molecular techniques including polymerase chain reaction (PCR) by amplification of the 16S rRNA gene. Furthermore, sequencing of the amplicons confirmed all ten isolates as Salmonella strains. The antigenic cross-reactivity was found maximum between the HB1 (strain isolated from honeybee) antiserum and its antigen with an antibody titer of 1:128, while the HB1 antiserum showed a cross-reactive titer range of 1:8 to 1:64. On the basis of the highest geometric mean titer (GMT) shown by the antiserum of the HB1 antigen, it was selected as the best candidate for a cross-reactive live Salmonella oral antigen. Moreover, the HB1 antigen was used a live oral antigen (1 × 1010 CFU/ml) in a safety test in rabbits and proved to be avirulent. During the animal trial, three different oral doses of the HB1 live oral antigen were evaluated in four different rabbits' groups (R1, R2, R3, and R4). The dose number 2 of 0.5 ml (two drops orally and repeated after one week) gave the best GMT measured by indirect hemagglutination (IHA) as compared to the other two doses, while R4 group was kept as control. Results of the challenge protection test also validated the efficacy of the double dose of the HB1 live vaccine, which gave the highest survival percentage. Results of this study lay the foundation for a potential cross-reactive live oral Salmonella vaccine that has proved to be immunogenic in rabbits.In this research, Salmonella species were isolated from the animal, insect and human enteric sources in Faisalabad, Punjab, Pakistan. These species were characterized by different microbiological and molecular techniques including polymerase chain reaction (PCR) by amplification of the 16S rRNA gene. Furthermore, sequencing of the amplicons confirmed all ten isolates as Salmonella strains. The antigenic cross-reactivity was found maximum between the HB1 (strain isolated from honeybee) antiserum and its antigen with an antibody titer of 1:128, while the HB1 antiserum showed a cross-reactive titer range of 1:8 to 1:64. On the basis of the highest geometric mean titer (GMT) shown by the antiserum of the HB1 antigen, it was selected as the best candidate for a cross-reactive live Salmonella oral antigen. Moreover, the HB1 antigen was used a live oral antigen (1 × 1010 CFU/ml) in a safety test in rabbits and proved to be avirulent. During the animal trial, three different oral doses of the HB1 live oral antigen were evaluated in four different rabbits’ groups (R1, R2, R3, and R4). The dose number 2 of 0.5 ml (two drops orally and repeated after one week) gave the best GMT measured by indirect hemagglutination (IHA) as compared to the other two doses, while R4 group was kept as control. Results of the challenge protection test also validated the efficacy of the double dose of the HB1 live vaccine, which gave the highest survival percentage. Results of this study lay the foundation for a potential cross-reactive live oral Salmonella vaccine that has proved to be immunogenic in rabbits.

Evaluation of a Salmonella Strain Isolated from Honeybee Gut as a Potential Live Oral Vaccine Against Lethal Infection of Salmonella Typhimurium

In this research, Salmonella species were isolated from the animal, insect and human enteric sources in Faisalabad, Punjab, Pakistan. These species were characterized by different microbiological and molecular techniques including polymerase chain reaction (PCR) by amplification of the 16S rRNA gene. Furthermore, sequencing of the amplicons confirmed all ten isolates as Salmonella strains. The antigenic cross-reactivity was found maximum between the HB1 (strain isolated from honeybee) antiserum and its antigen with an antibody titer of 1:128, while the HB1 antiserum showed a cross-reactive titer range of 1:8 to 1:64. On the basis of the highest geometric mean titer (GMT) shown by the antiserum of the HB1 antigen, it was selected as the best candidate for a cross-reactive live Salmonella oral antigen. Moreover, the HB1 antigen was used a live oral antigen (1 × 1010 CFU/ml) in a safety test in rabbits and proved to be avirulent. During the animal trial, three different oral doses of the HB1 live oral antigen were evaluated in four different rabbits’ groups (R1, R2, R3, and R4). The dose number 2 of 0.5 ml (two drops orally and repeated after one week) gave the best GMT measured by indirect hemagglutination (IHA) as compared to the other two doses, while R4 group was kept as control. Results of the challenge protection test also validated the efficacy of the double dose of the HB1 live vaccine, which gave the highest survival percentage. Results of this study lay the foundation for a potential cross-reactive live oral Salmonella vaccine that has proved to be immunogenic in rabbits.

DNA Diagnostics for Schistosomiasis Control.

Despite extensive efforts over the last few decades, the global disease burden of schistosomiasis still remains unacceptably high. This could partly be attributed to the lack of accurate diagnostic tools for detecting human and animal schistosome infections in endemic areas. In low transmission and low prevalence areas where schistosomiasis elimination is targeted, case detection requires a test that is highly sensitive. Diagnostic tests with low sensitivity will miss individuals with low infection intensity and these will continue to contribute to transmission, thereby interfering with the efficacy of the control measures operating. Of the many diagnostic approaches undertaken to date, the detection of schistosome DNA using DNA amplification techniques including polymerase chain reaction (PCR) provide valuable adjuncts to more conventional microscopic and serological methods, due their accuracy, high sensitivity, and the capacity to detect early pre-patent infections. Furthermore, DNA-based methods represent important screening tools, particularly in those endemic areas with ongoing control where infection prevalence and intensity have been reduced to very low levels. Here we review the role of DNA diagnostics in the path towards the control and elimination of schistosomiasis.

Identification of a mitochondrial 12S rRNA A1555G mutation causing aminoglycoside-induced deafness in a large Thai family

Abstract Background Hearing loss is among the most frequent sensory disorders. Preventable causes include medications given to genetically susceptible individuals. Several families around the world with an A1555G mitochondrial mutation who became profoundly deaf after receiving aminoglycosides have been described. However, none has been reported in Thailand. Objectives To identify the cause of hearing loss of a large Thai family with 11 members who reportedly turned deaf after receiving antibiotics. Methods We obtained blood samples from 5 members; 4 of whom had hearing loss. Mutation analyses were performed using molecular techniques including polymerase chain reaction, Sanger sequencing, and restriction fragment length polymorphism. Results All 4 affected members were found to harbor the same A1555G mitochondrial mutation, while the unaffected had only the wild-type A. Conclusions We have identified the mitochondrial mutation leading to aminoglycoside-induced hearing loss in a Thai population. Raising awareness for medical practitioners of this genetic susceptibility in Thailand is warranted. Avoidance of certain medications in these individuals would prevent this acquired permanent hearing loss.

Utility of polymerase chain reaction for detection of Mycobacterium tuberculosis in suspected cases of tuberculosis lymphadenopathy

Introduction: There is a need for a rapid and cost-effective technique for reliable diagnosis of tubercular lymphadenopathy, particularly in low-resource setting. In this study, we have used various diagnostic techniques including polymerase chain reaction (PCR) to diagnose clinically suspected cases of tubercular lymphadenopathy and compared the results to see which of the techniques are more sensitive, specific, and cost-effective. Materials and Methods: All patients having swelling in the neck, axillary, and inguinal regions were recruited for the study. Sputum for acid-fast Bacillus (AFB), fine-needle aspiration cytology, excision biopsy, DNA-PCR, AFB smear of the same material was done as per standard protocol. Results: 32% patients have granulomas with necrosis, whereas 30% have acute suppurative lesions and 24% and 14% patients were having only granulomas and only necrosis, respectively. A significant difference was observed between the PCR-negative and positive cases with respect to their cytomorphologic features. Positive AFB and tuberculosis-PCR (TB-PCR) results were significantly more common in the cases with chronic granulomatous inflammation in comparison to the cases showing chronic inflammation only. Sensitivity and specificity of TB-PCR were found to be 71.4% and 28.4%, respectively. Conclusion: PCR is a sensitive and rapid technique in the demonstration of Mycobacterium tuberculosis . It should be done in clinically suspected patients of tuberculous lymphadenopathy when their AFB stain is even negative.

Integrated electrochemical microsystems for genetic detection of pathogens at the point of care.

The capacity to achieve rapid, sensitive, specific, quantitative, and multiplexed genetic detection of pathogens via a robust, portable, point-of-care platform could transform many diagnostic applications. And while contemporary technologies have yet to effectively achieve this goal, the advent of microfluidics provides a potentially viable approach to this end by enabling the integration of sophisticated multistep biochemical assays (e.g., sample preparation, genetic amplification, and quantitative detection) in a monolithic, portable device from relatively small biological samples. Integrated electrochemical sensors offer a particularly promising solution to genetic detection because they do not require optical instrumentation and are readily compatible with both integrated circuit and microfluidic technologies. Nevertheless, the development of generalizable microfluidic electrochemical platforms that integrate sample preparation and amplification as well as quantitative and multiplexed detection remains a challenging and unsolved technical problem. Recognizing this unmet need, we have developed a series of microfluidic electrochemical DNA sensors that have progressively evolved to encompass each of these critical functionalities. For DNA detection, our platforms employ label-free, single-step, and sequence-specific electrochemical DNA (E-DNA) sensors, in which an electrode-bound, redox-reporter-modified DNA "probe" generates a current change after undergoing a hybridization-induced conformational change. After successfully integrating E-DNA sensors into a microfluidic chip format, we subsequently incorporated on-chip genetic amplification techniques including polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) to enable genetic detection at clinically relevant target concentrations. To maximize the potential point-of-care utility of our platforms, we have further integrated sample preparation via immunomagnetic separation, which allowed the detection of influenza virus directly from throat swabs and developed strategies for the multiplexed detection of related bacterial strains from the blood of septic mice. Finally, we developed an alternative electrochemical detection platform based on real-time LAMP, which not is only capable of detecting across a broad dynamic range of target concentrations, but also greatly simplifies quantitative measurement of nucleic acids. These efforts represent considerable progress toward the development of a true sample-in-answer-out platform for genetic detection of pathogens at the point of care. Given the many advantages of these systems, and the growing interest and innovative contributions from researchers in this field, we are optimistic that iterations of these systems will arrive in clinical settings in the foreseeable future.

Detection of epidermal growth factor receptor (EGFR) exon 19 E746-A750 deletion and EGFR exon 21 point mutations in lung adenocarcinoma by immunohistochemistry: a comparative study to EGFR exons 19 and 21 mutations analysis using PCR followed by high-resolution melting and pyrosequencing

Activating mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC) with adenocarcinoma histology confers susceptibility to treatment with EGFR tyrosine kinase inhibitors (TKI). Analysis of these activating mutations is typically performed by molecular techniques including polymerase chain reaction (PCR) amplification followed by deoxyribonucleic acid (DNA) melting curve analysis and/or direct sequencing. Recently developed mutation-specific immunohistochemical stains for the L858R point mutation in exon 21 and an E746-A750 deletion in exon 19 of the EGFR gene may offer a fast, cost-effective alternative to molecular testing. In this study, EGFR exons 19 and 21 mutations were analyzed using EGFR E746-A750 del and EGFR L585R point mutation-specific antibodies and the results were compared with the exon 19 deletions and exon 21 L588R point mutation as detected by molecular testing. The findings demonstrate a high concordance rate (100% in 12 cases) between the two methodologies for EGFR exon 21 L585R point mutation but a low concordance rate (54% in 22 cases) for exon 19 deletions when using EGFR E746-A750 mutation-specific antibody compared to molecular testing. This low concordance rate between the two methods is due to the fact that EGFR exon 19 mutations include deletions other than E746-A750 as well as insertion/deletions that will not be covered by exon 19 E746-A750 del mutation-specific antibody. Despite this limitation, the current EGFR mutation-specific antibodies can prove useful as an alternative to molecular tests for exon 21 L585R mutations as well as a subset of EGFR exon 19 deletion mutations, particularly in cases with low percentage of tumor concentration and in decalcified tissue specimens that are not suitable for DNA-based molecular assays.

Bacterial source tracking guides management of boat head waste in a coastal resort area

Fecal contamination of water bodies causes a public health problem and economic loss. To control such contamination management actions need to be guided by sound science. From 2007–2009 a study was undertaken to determine the sources of fecal bacteria contamination to the marine waters adjoining the Town of Wrightsville Beach, North Carolina, USA. The research effort included sampling for fecal coliform and Enterococcus bacteria, sampling for optical brighteners, dye studies, and use of molecular bacterial source tracking techniques including polymerase chain reaction (PCR) and terminal restriction fragment polymorphism (T-RFLP) fingerprinting of the Bacteroides–Prevotella group. Of the 96 samples collected from nine locations during the study, the water contact standard for Enterococcus was exceeded on 13 occasions. The T-RFLP fingerprint analyses demonstrated that the most widespread source of fecal contamination was human, occurring in 38% of the samples, with secondary ruminant and avian sources also detected. Optical brightener concentrations were low, reflecting a lack of sewage line leakage or spills. A lack of sewer leaks and lack of septic systems in the town pointed toward discharge from boat heads into the marine waters as the major cause of fecal contamination; this was supported by dye studies. Based on these data, the Town initiated action to have the U.S. Environmental Protection Agency declare the coastal waters (out to 3 nautical miles), the nearby Atlantic Intracoastal Waterway and its tributaries a no-discharge zone (NDZ) to alleviate the human fecal pollution. The Town garnered supporting resolutions from other local communities who jointly petitioned the North Carolina Department of Environmental and Natural Resources. This State regulatory agency supported the local government resolutions and sent an application for an NDZ to the EPA in April 2009. The EPA concurred, and in February 2010 the coastal waters of New Hanover County, NC, became the first marine area on the U.S. eastern seaboard between Delaware and the Florida Keys to be declared a no-discharge zone.

The Etiology, Pathophysiology, and Management of Otitis Media with Effusion.

Otitis media with effusion (OME) is a common and important condition that may result in developmental delay in children, and significant health care resources are devoted to its management. Newer techniques including polymerase chain reaction are implicating organisms not previously considered important in etiology. The role of gastroesophageal reflux as a cause of OME is likely to receive greater research attention. Regarding prevention, more is being learned about potentially modifiable risk factors such as environmental smoke, care outside the home, and breast feeding. Although immunization may to play a role in the future, existing evidence suggests that the general population of children should not be immunized in order to prevent OME. Several major studies have recently added to the understanding of epidemiology and management. Large trials in the United States, the Netherlands, and the UK suggest that OME is not an appropriate condition to include in a screening program. In addition, the advantages of early treatment with ventilation tubes over watchful waiting in terms of language development tend be modest and diminish by about 18 months. Treatment with hearing aids should be further evaluated. The search for effective medical management continues, and better ways are being identified of targeting interventions to those children with OME who are most likely to benefit.

Restriction enzyme mapping of bacterial urease genes: using degenerate primers to expand experimental outcomes

AbstractTwo degenerate primers are described that can be used to amplify a 340 bp fragment of the ureC gene from a variety of urease producing bacteria. A series of experiments are outlined that enable students to develop restriction maps of the urease gene clusters from different bacteria. Students learn a variety of molecular biology techniques including polymerase chain reaction, agarose gel electrophoresis, and Southern blotting. This project allows for multiple experimental outcomes using the same two PCR primers. © 2001 IUBMB. Published by Elsevier Science Ltd. All rights reserved.

Restriction enzyme mapping of bacterial urease genes: using degenerate primers to expand experimental outcomes

Two degenerate primers are described that can be used to amplify a 340bp fragment of the ureC gene from a variety of urease producing bacteria. A series of experiments are outlined that enable students to develop restriction maps of the urease gene clusters from different bacteria. Students learn a variety of molecular biology techniques including polymerase chain reaction, agarose gel electrophoresis, and Southern blotting. This project allows for multiple experimental outcomes using the same two PCR primers.

Serine Proteases in Rodent Hippocampus

Brain serine proteases are implicated in developmental processes, synaptic plasticity, and in disorders including Alzheimer's disease. The spectrum of the major enzymes expressed in brain has not been established previously. We now present a systematic study of the serine proteases expressed in adult rat and mouse hippocampus. Using a combination of techniques including polymerase chain reaction amplification and Northern blotting we show that tissue-type plasminogen activator (t-PA) is the major species represented. Unexpectedly, the next most abundant species were RNK-Met-1, a lymphocyte protease not reported previously in brain, and two new family members, BSP1 (brain serine protease 1) and BSP2. We report full-length sequences of the two new proteases; homologies indicate that these are of tryptic specificity. Although BSP2 is expressed in several brain regions, BSP1 expression is strikingly restricted to hippocampus. Other enzymes represented, but at lower levels, included elastase IV, proteinase 3, complement C2, chymotrypsin B, chymotrypsin-like protein, and Hageman factor. Although thrombin and urokinase-type plasminogen activator were not detected in the primary screen, low level expression was confirmed using specific polymerase chain reaction primers. In contrast, and despite robust expression of t-PA, the usual t-PA substrate plasminogen was not expressed at detectable levels.

The epidemiology of myelopathy associated with human T-lymphotropic virus 1

A progressive spastic myelopathy is one of the principal manifestations of the human T cell lymphotropic virus type 1. Recent research is reviewed on the geography and epidemiology of this specific form of tropical spastic paraparesis. First recognized in the Caribbean, Colombia and Japan, it is now also confirmed as a major neurological problem in areas of eastern Brazil, western equatorial Africa, Natal and Seychelles, with other scattered foci world-wide. Accurate surveys call for sophisticated techniques including polymerase chain reaction amplification. The only defined modes of spread are by breast feeding, sexual contact and blood cell infusion. The onset of neurological disease is associated with high antibody titres and a high rate of spontaneous T lymphocyte proliferation. Molecular analysis has revealed no nucleotide sequence variation in cases with or without myelopathy. In non-transfusion cases the clinical attack rate is low with a very long latent period, but there are unexplained regional differences and familial cases are an important exception. Current research is focused on possible local, ethnic, or genetic co-factors.