PCR-free blood group genotyping using a nanobiosensor.

BACKGROUND AND OBJECTIVES: The last two decades have seen major developments in genotyping assays to facilitate the procurement of red blood cell units to alloimmunized patients. To make genotyping faster, simpler and less costly, a nanotechnology approach based on metal/silica fluorescent nanoparticles and a polymer-based hybridization optical transducer was designed. The objectives of this study were (1) to verify whether this nanobiosensor has the ability to discriminate single nucleotide polymorphisms in non-amplified genomic DNA and (2) to establish whether the signal generated by the nanobiosensor is sufficiently intense to be detected by standard flow cytometry. MATERIALS AND METHODS: Silver-core silica-shell fluorescent nanoparticles (Ag@SiO2) were prepared, and amine-modified DNA probes were grafted to their surface. A cationic conjugated polymer was electrostatically bound to the surface probes to become optically active upon hybridization with a target. Two nanobiosensor formulations specific to DO*01 and DO*02 alleles were prepared. DNA was extracted from whole blood and mixed with the nanobiosensor for hybridization. The nanobiosensor fluorescence was measured by flow cytometry. RESULTS: Nine volunteers were typed for Dombrock blood group antigens DO*01 and DO*02. A statistically significant increase in the optical transduction signal was observed for sequence-specific samples. All nine genotypes were correctly identified when compared to standardized PCR assays. CONCLUSION: The nanobiosensor provides rapid and simple genotyping of blood group antigens from unamplified genomic DNA and can be measured using standard flow cytometers. This PCR-free approach could be applied to any known genetic polymorphism.

Red blood cell antigen portrait of self-identified black donors in Quebec.

OBJECTIVES: The goal of this study was to establish a red blood cell antigen portrait of self-identified Black donors for the province of Quebec, Canada. BACKGROUND: The demand for extensively phenotyped red blood cells is on the rise. A good example is the sickle cell patient cohort. To better answer their transfusion needs, Héma-Québec put forward great efforts to increase the recruitment of donors among cultural communities. MATERIALS AND METHODS: In October 2009, an optional question was added on the record of donation to indicate the donor's ethnicity. Self-identified Black donors were extensively phenotyped by the Immunohematology Laboratory, whereas the Research and Development team genotyped red blood cell antigens to complete the picture. RESULTS: Approximately 1500 self-identified Black donors have donated blood at least once since the beginning of the programme. Genotyping results predicted rare phenotypes: 18 S-s- (3 U-, 15 U+(w) ), 15 Js(a+b-), 5 Hy-, 3 Jo(a-), 34 hr(B) +(w) /- and 15 hr(B)-. CONCLUSION: These Black donors, with or without a rare phenotype, are precious to the patient cohort depending on blood transfusions and to our organisation as the blood provider for the whole province of Quebec.

A new SEMA7A variant found in Native Americans with alloantibody.

BACKGROUND AND OBJECTIVES: John Milton Hagen (JMH) antigens are carried by Semaphorin 7A that plays important roles in the nervous system and the immune responses. Its role on the erythrocytes is unclear. Over the years, few samples were referred to our Immunohaematology Reference Laboratory to elucidate their JMH status. MATERIALS AND METHODS: Seven blood samples with antibodies compatible with JMH1-negative red cells were studied at the molecular level to identify polymorphisms and explain the JMH diversity observed. Four samples were of Native American background and three were Caucasians. Molecular analyses of the SEMA7A were undertaken, and soluble form of recombinant Sema7A proteins was produced to characterize the antibodies. RESULTS: Sequencing of the cDNA showed a polymorphism in SEMA7A exon 9 at position 1040 (G>T) in the four Native American samples. Caucasians had a normal sequence. This polymorphism precludes a change at position 347 where an Arg is replaced by a Leu. Plasma was assayed in ELISA on wild-type Sema7AArg347 and variant Sema7ALeu347 proteins. Results clearly indicated a specific recognition of the antibody produced by the Native Americans for the wild-type Sema7AArg347 protein and not the variant one. CONCLUSION: A new SEMA7A variant was identified in this study. The antibody present in the Native American plasma samples should be considered as an alloantibody because it recognizes the wild-type protein.

Consortium for Blood Group Genes (CBGG): 2009 report.

Consortium for Blood Group Genes is a worldwide organization whose goal is to have a vehicle to interact, establish guidelines, operate a proficiency program, and provide education for laboratories involved in DNA and RNA testing for the prediction of blood group, platelet, and neutrophil antigens. Currently, the consortium operates with representatives from Brazil, Canada, and the United States. Membership is voluntary with the expectation that members actively contribute to discussions involving blood group genetics. This year witnessed a change in the standing committee membership and the institution of a representative for the human platelet antigens group. Looking forward, the consortium sees challenges for the nomenclature of blood group alleles and user-required specifications for laboratory information systems to store genotype information.

[Genotyping of 21,000 blood donors in Quebec and RHD analysis]. / Génotypage de 21 000 donneurs de sang au Québec et analyse du RHD.

STUDY GOAL: A repeat blood donor genotyping project was launched by Héma-Québec in October 2007. The objective was to screen 21,000 samples for 22 polymorphisms for red blood cell and platelet blood groups to build a database to easily find compatible donors. MATERIALS AND METHODS: Donors who have donated at least three times during the last year were selected. A drop of blood was spotted on FTA paper and sent to the Pharmacogenomic Centre at the Montreal Heart Institute for analysis. All genotype results were compared to the known phenotype. In parallel, the RHD gene of D negative blood donors was examined. RESULTS: Less than two years were necessary to complete the database. The genotype/phenotype concordance was 99.6% with only 165 discrepancies observed and further analysed. More than 55% of these discrepancies confirmed the initial genotype. The RHD study done on D negative samples found 13 donors positive for a variant RHD gene. Four were RHD*Ψ positive, while the other nine presented variant polymorphisms precluding a reduced expression of the D antigen. CONCLUSION: Thanks to this project, Héma-Québec is able to answer increasing demands for compatible blood more rapidly. The organisation has also demonstrated the security of its D negative inventory.

Set-up and routine use of a database of 10,555 genotyped blood donors to facilitate the screening of compatible blood components for alloimmunized patients.

BACKGROUND AND OBJECTIVES: Large-scale genotyping of blood donors for red blood cell and platelet antigens has been predicted to replace phenotyping assays in the screening of compatible blood components for alloimmunized patients. Although several genotyping platforms have been described, novel procedures and processes are needed to perform genotyping efficiently and to maximize its benefits for blood banks. MATERIALS AND METHODS: Here we describe the processes and procedures developed to introduce large-scale genotyping in our routine operations. RESULTS: Preliminary cost-benefit analysis indicated that genotyping must target frequent blood donors (> 3 donations/year) to be efficiently used. A custom-designed computer application was developed to manage the whole project. It selects frequent donors among recent donations, prints coded labels to identify blood samples sent to the external genotyping laboratory, and stores genotyping results. It can search for donors compatible for any combination of the 22 genotyped antigens as well as consult the current inventory for the presence of the corresponding blood components. The phenotype of recovered components is confirmed by standard serology techniques prior to shipment to hospitals. CONCLUSION: Since October 2007, 10 555 blood donors have been genotyped. The database is used on a regular basis to find compatible blood components with a genotype-phenotype concordance of 99.6%.

Bovine immunodeficiency virus: identification of a long terminal repeat sequence with enhanced promoter activity.

We previously identified a new bovine immunodeficiency virus (BIV) trans-activator factor of transcription (Tat236) that was derived from a variant of BIV. Here, we report a new BIV long terminal repeat (LTR) sequence (LTRn) that was obtained by PCR from the DNA of cells infected with the BIV variant mentioned above. Sequence analysis indicated that the LTRn U3 region harbors three nucleic acid mutations at residue positions -194, -135 and -114 when compared to the original (wild-type) LTR sequence. Reporter gene assays indicated that LTRn promotes basal and Tat-mediated transactivation activity to levels significantly higher than those obtained with the wild-type LTR. Restoration experiments to the wild-type genotype indicated that both the -135 and -114 nucleic acid substitutions were responsible for the enhanced promoter activity of BIV LTRn.

Sensory neuron and substance P involvement in symptoms of a zymosan-induced rat model of acute bowel inflammation.

Intestinal inflammation is a painful syndrome with multiple symptoms, including chronic pain. This study examined the possible role of sensory neurons and substance P in symptoms of an animal model of acute intestinal inflammation. The model was induced by injecting ethanol and zymosan into the colon of anesthetized male rats. Three hours later, sections of the colon were stained with hematoxylin and eosin. To determine the role of substance P, 5 mg/kg of the neurokinin-1 receptor (NK-1r) antagonist, CP-96,345, or 300 microg/kg of an antisense oligonucleotide targeted at NK-1r mRNA was administered. Spinal cord sections were examined for internalization of NK-1r, as an indicator of substance P release. Sections of colon revealed infiltration of inflammatory cells following ethanol and zymosan treatment. Plasma extravasation in rats given ethanol and zymosan was significantly greater than in controls given saline only (P<0.0001) or saline and ethanol (P<0.001). In ethanol- and zymosan-treated rats given CP-96,345, plasma extravasation was significantly less than in rats given ethanol and zymosan without the antagonist (P<0.0001). Administration of the antisense oligonucleotide also resulted in lower levels of plasma extravasation compared with controls (P<0.01). Internalization of the NK-1r was observed in neurons of lamina I in the T13-L2 and L6-S2 regions of the spinal cord, as well as in sympathetic preganglionic neurons at the L1 level. This internalization was observed in the absence of any other stimulus besides the inflammation itself. This study implicates substance P and its receptor, the NK-1r, in acute inflammation of the colon.

HIV prevalence and trends from data in Zimbabwe, 1997-2004.

BACKGROUND: This paper brings together data from a variety of reports to provide a basis for assessing future steps for responding to and monitoring the HIV epidemic in Zimbabwe. METHOD: Data reported from four antenatal clinic (ANC) surveys conducted between 2000 and 2004, two small local studies in Zimbabwe conducted from 1997 through 2003, four general population surveys from 1999 through 2003, and service statistics covering 1990 through 2004 were used to describe recent trends in HIV prevalence and incidence, behaviour change, and programme provision. RESULTS: HIV prevalence among pregnant women attending ANCs declined substantially from 32.1% in 2000 to 23.9% in 2004. The local studies confirmed the decline in prevalence. However, prevalence continued to be high. Sexual behaviour data from surveys suggests a reduction in sexual experience before age 15 years among both males and females age 15-19 years, and in the proportions of males and females aged 15-29 years reporting non-regular sexual partners in the past 12 months. Reported condom use with non-regular partners has been high since 1999. Condom distribution and HIV counseling and testing increased from 2000 to 2004. DISCUSSION: On the basis of examination of data from a variety of sources, the recent decrease in HIV prevalence may be related to recent reductions in early-age sexual activity and non-regular sexual partnerships and increases in condom use. Comparison of data from sentinel surveillance systems, population based serosurveys, local studies, and service statistics provide increased confidence that a decline in HIV prevalence in Zimbabwe is actually happening in the population.

Postnatal development of ectopic sensory fibers containing endomorphin-2 in the white matter of the spinal cord of a transgenic mouse expressing nerve growth factor in oligodendrocytes.

Transgenic mice ectopically expressing nerve growth factor in oligodendrocytes have high levels of nerve growth factor immunoreactivity in the white matter of the spinal cord from birth until 2 months of age. The nerve growth factor over-expression leads to the appearance of ectopic substance P containing sensory fibers in the white matter of the spinal cord that persist throughout the life of the animal. These transgenic mice have been found to display hypersensitivity to a thermal stimulus following a sensitizing pinch stimulus known to release endogenous substance P. Surprisingly, this hypersensitivity is completely reversed following the administration of morphine, to the extent that transgenic mice become less sensitive to pain than the wild type mice given morphine. Endomorphin-2, an endogenous opioid peptide, has been found co-localized with substance P in primary sensory fibers in the spinal cord. In this study, we show that the ectopic fibers also express endomorphin-2, and describe the postnatal development of such expression, as detected by immunocytochemistry. We confirmed that endomorphin-2 expression starts later in the postnatal period than substance P. Surprisingly, transgenic animals had delayed appearance of endomorphin-2 in the superficial dorsal horn, compared with wild type, and expressed particularly high levels of endomorphin-2 immunoreactivity in the ectopic fibers from postnatal days 10-30, coinciding with the peak of nerve growth factor expression in oligodendrocytes. Endomorphin-2 immunoreactivity was still readily detected in ectopic fibers of 120-day-old animals. Furthermore, we detected immunoreactivity for the mu-opioid receptor in the ectopic fibers, where it was co-localized with endomorphin-2 immunoreactivity. In the superficial dorsal horn, there were no apparent differences in the distribution and intensity of mu-opioid receptor immunoreactivity between wild type and transgenic animals. Taken together, these data could provide an explanation for the enhanced effect of opioid analgesics in transgenic mice, when compared with control mice, as well as provide the basis for studies of the postnatal development of the hyperalgesia and allodynia demonstrated by these animals.

The bovine immunodeficiency virus: cloning of a tat/rev cDNA encoding a novel Tat protein with enhanced transactivation activity.

Previous studies have shown that BIV may encode two types of Tat proteins of 103 and 108 amino acids, respectively. Here, we report the characterization of a new BIV Tat protein (Tat236) derived from a tat/rev cDNA. The tat/rev cDNA was obtained by reverse transcription-PCR from RNA extracted from cells infected with BIV. BIV was rescued by cell co-cultivation from the spleen of rabbits exposed for 3 years to the R29 isolate of BIV. Sequence analysis indicated that BIV Tat236 contains the first 98 amino acids of Tat103 and the 3' end 138 amino acids of Rev. Reporter gene assays indicated that transactivation of BIV long terminal repeat (LTR) by Tat236 is higher than by the original BIV Tat proteins in several cell types. By using overlapping deletion mutants, evidence was given that the predicted basic domain of Rev within Tat236 plays a major role in the observed enhanced transactivation activity of the protein. However, the intact functional domain of the original BIV Tat is required for efficient transactivation. This is the first report of a hybrid Tat protein from BIV or any lentiviruses that shows higher transactivation than the original transactivator Tat proteins.

Leaving sex work: barriers, facilitating factors and consequences for female sex workers in northern Thailand.

Factors facilitating or inhibiting women's ability to leave sex work are still poorly characterized, and little is known about women's lives after they leave the profession. This paper presents findings from a qualitative study about factors affecting women's ability to leave sex work and influencing their lives after leaving. We interviewed 42 current and former female sex workers (FSWs) drawn from a cohort study of 500 FSWs in northern Thailand. All but one of the participants had quit sex work at least once. The majority experienced one or more quit-re-entry-quit cycles. Women's ability and decisions to leave sex work were determined primarily by four factors: economic situation, relationship with a steady partner, attitudes towards sex work and HIV/AIDS experience. Economic concerns, ranging from survival needs to materialistic desires, had the strongest influence. Most women perceived their risk for HIV infection to be lower after leaving sex work, but three of the 17 HIV-infected women acquired infection after having left, presumably from their steady partners. Prevention efforts should guide women as they transition out of commercial sex work. Interventions aimed at assisting women wanting to leave sex work need to address the role of economic factors.

An outbreak of syphilis in Alabama prisons: correctional health policy and communicable disease control.

OBJECTIVES: After syphilis outbreaks were reported at 3 Alabama State men's prisons in early 1999, we conducted an investigation to evaluate risk factors for syphilis infection and describe patterns of syphilis transmission. METHODS: We reviewed medical, patient interview, and prison transfer records and documented sexual networks. Presumptive source cases were identified. Odds of exposure to unscreened jail populations and transfer from other prisons were calculated for case patients at 1 prison. RESULTS: Thirty-nine case patients with early syphilis were identified from 3 prisons. Recent jail exposure (odds ratio [OR] = 8.0, 95% confidence interval [CI] = 0.3, 158.7, P = .14) and prison transfer (OR = 32.0, 95% CI = 1.6, 1668.1, P < .01) were associated with being a source case patient. CONCLUSIONS: Probable sources of syphilis introduction into and transmission within prisons included mixing of prisoners with unscreened jail populations, transfer of infected inmates between prisons, and multiple concurrent sexual partnerships. Reducing sexual transmission of disease in correctional settings is a public health priority and will require innovative prevention strategies.

Prevalence of genital chlamydial infection in young women entering a national job training program, 1990-1997.

OBJECTIVES: This analysis describes trends in the prevalence of genital chlamydial infection in economically disadvantaged young women entering a national job training program. METHODS: We examined chlamydia test data for May 1990 through June 1997 for women aged 16 to 24 years who enrolled in the program. The significance of trends was evaluated with the chi 2 test for trend. RESULTS: Prevalence of chlamydial infection declined 32.9%, from 14.9% in 1990 to 10.0% in 1997 (P < .001). Prevalence decreased significantly in all age groups, racial/ethnic groups, and geographic regions. CONCLUSIONS: The decrease in prevalence of chlamydial infection suggests that prevention activities have reached disadvantaged women across the United States; however, prevalence of chlamydial infection remains high, and enhanced prevention efforts in disadvantaged communities are urgently needed.

Characteristics of persons with syphilis in areas of persisting syphilis in the United States: sustained transmission associated with concurrent partnerships.

BACKGROUND AND GOAL: In areas with persistent syphilis, to characterize persons at higher risk for transmitting syphilis. STUDY DESIGN: Cohort study. Structured interviews of persons with early syphilis from four research centers were linked to outcomes of partner tracing. RESULTS: Of 743 persons with syphilis, 229 (31%) reported two or more partners in the previous month, and 57 (8%) received money or drugs for sex in the previous three months. Persons with at least one partner at an earlier stage of syphilis than themselves were defined as transmitters; 63 (8.5%) of persons with early syphilis met this definition. Having concurrent partners (two or more in one week in the last month) was independently associated with being a transmitter. CONCLUSION: Sexual network/behavioral characteristics of syphilis patients and their partners, such as concurrency, can help identify persons at higher risk for transmitting syphilis who should receive emphasis in disease prevention activities.

Syphilis among HIV-infected mothers and their infants in Texas from 1988 to 1994.

BACKGROUND: Syphilis was investigated in a group of HIV-infected women and their infants. GOAL: To assess syphilis morbidity among HIV-infected women and their infants. Among women with syphilis during pregnancy, the risks for delivering an infant with congenital syphilis were assessed. STUDY DESIGN: Through the Pediatric Spectrum of HIV Disease project, Texas infants born to HIV-infected women were identified. After the infants were matched with their mothers, it was determined which had been reported as syphilis cases. RESULTS: In this study 18% of the HIV-infected mothers were reported as syphilis cases, most during pregnancy. Half of these mothers delivered infants (n = 49) with congenital syphilis. Inadequate prenatal care was the only significant risk for delivering an infant with congenital syphilis. The congenital syphilis rate among Texas infants of HIV-infected mothers was 48.8 per 1,000 live births. CONCLUSION: The congenital syphilis rate among Texas infants born to HIV-infected mothers was almost 50 times that of the general population.

Interleukin 12 secretion enhances antitumor efficacy of oncolytic herpes simplex viral therapy for colorectal cancer.

OBJECTIVE: To assess the strategy of combining oncolytic herpes simplex virus (HSV) therapy with immunomodulatory therapy as treatment for experimental colon cancer. The oncolytic HSV recombinant NV1023 and the interleukin 12 (IL-12)-secreting oncolytic NV1042 virus were evaluated in vitro and in vivo with respect to antitumor efficacy. SUMMARY BACKGROUND DATA: Genetically engineered, replication-conditional, attenuated HSVs have shown oncolytic activity against a wide variety of solid malignancies. Other strategies for treating cancer have involved immunomodulation and cytokine gene transfer using viral vectors. This study has combined both of these strategies by inserting the murine IL-12 gene into a replication-competent HSV. This approach allows oncolytic therapy to replicate selectively within and lyse tumor cells while providing the host immune system with the cytokine stimulus necessary to recruit and activate inflammatory cells needed to enhance the antitumor effect. METHODS: NV1023 is a multimutant HSV based on the wild-type HSV-1 F strain. NV1042 was created by insertion of the mIL-12 gene into NV1023. Cytotoxicity and viral proliferation of both NV1023 and NV1042 within murine CT26 colorectal cancer cells were first shown. Cells infected with NV1042 were then shown to produce significant levels of IL-12. Using an experimental flank model of colon cancer, mice were treated with both high and low doses of NV1023 or NV1042 and were followed up for both cure and reduction in tumor burden. RESULTS: Both viruses could replicate within and kill CT26 cells in vitro, with 100% cytotoxicity achieved after infection by either virus. Only NV1042 could produce mIL-12. Therapy using high viral doses to treat animals in vivo showed equal efficacy between NV1023 and NV1042, with five of seven cures for each virus. When viral doses were lowered, only the cytokine-producing NV1042 virus could reduce tumor burden and cure animals of their disease. CONCLUSIONS: Both NV1023 and NV1042 have the oncolytic potential to kill colon cancer cells at higher doses. Cytokine production by NV1042 may allow lower doses of viral therapy to be used without losing antitumor efficacy. The combination of oncolytic viral therapy and immunomodulatory strategies should be further investigated as treatment for colon cancer.

Sexually transmitted diseases of older persons in Washington State.

BACKGROUND: Sexually transmitted diseases (STDs) in persons older than 50 years are rarely studied because STDs are more common in young people. Understanding the epidemiology of STDs in older persons is important for reducing STD morbidity and for improving STD care. GOAL: To understand the epidemiology of STDs in older persons. METHODS: Washington State's STD surveillance data from 1992 to 1998 were analyzed to describe the burden of STDs and source of care for these diseases in older persons. RESULTS: From 1992 to 1998, 1535 episodes of STDs were reported for 50- to 80-year-olds in Washington State, accounting for 1.3% of all reported STDs. The most common STDs were nongonococcal urethritis in men and genital herpes in women. As compared with younger persons, older individuals more frequently sought care at private clinics and had symptoms at the time of the clinic visit. CONCLUSIONS: Sexually transmitted diseases are reported among older persons, although at lower rates than among younger persons. Services for STD and counseling regarding safe sex should be available to persons of all ages.

Molecular subtyping of Treponema pallidum in an Arizona County with increasing syphilis morbidity: use of specimens from ulcers and blood.

A molecular-based subtyping system for Treponema pallidum was used during an investigation of increasing syphilis in Maricopa County, Arizona. Genital ulcer or whole blood specimens from patients with syphilis were assayed by a polymerase chain reaction (PCR) amplification of a T. pallidum DNA polymerase I gene. Positive specimens were typed on the basis of PCR amplification of 2 variable genes. In all, 41 (93%) of 44 of ulcer specimens and 4 (27%) of 15 blood specimens yielded typeable T. pallidum DNA. Twenty-four (53%) of 45 specimens were subtype 14f; other subtypes identified included 4f, 4i, 5f, 12a, 12f, 14a, 14d, 14e, and 14i. Only 2 specimens were from epidemiologically linked patients. This investigation demonstrates that multiple subtypes of T. pallidum can be found in an area with high syphilis morbidity, although 1 subtype (14f) was predominant. Four typeable specimens were from blood, a newly identified specimen source for subtyping.