Prevalence of antibodies to hepatitis B, hepatitis C, and HIV and risk factors in entrants to Irish prisons: a national cross sectional survey.

OBJECTIVES: To determine the prevalence of antibodies to hepatitis B core antigen, hepatitis C virus, and HIV in entrants to Irish prisons and to examine risk factors for infection. DESIGN: Cross sectional, anonymous survey, with self completed risk factor questionnaire and oral fluid specimen for antibody testing. SETTING: Five of seven committal prisons in the Republic of Ireland. PARTICIPANTS: 607 of the 718 consecutive prison entrants from 6 April to 1 May 1999. MAIN OUTCOME MEASURES: Prevalence of antibodies to hepatitis B core antigen, hepatitis C virus, and HIV in prison entrants, and self reported risk factor status. RESULTS: Prevalence of antibodies to hepatitis B core antigen was 37/596 (6%; 95% confidence interval 4% to 9%), to hepatitis C virus was 130/596 (22%; 19% to 25%), and to HIV was 12/596 (2%; 1% to 4%). A third of the respondents had never previously been in prison; these had the lowest prevalence of antibodies to hepatitis B core antigen (4/197, 2%), to hepatitis C (6/197, 3%), and to HIV (0/197). In total 29% of respondents (173/593) reported ever injecting drugs, but only 7% (14/197) of those entering prison for the first time reported doing so compared with 40% (157/394) of those previously in prison. Use of injected drugs was the most important predictor of antibodies to hepatitis B core antigen and hepatitis C virus. CONCLUSIONS: Use of injected drugs and infection with hepatitis C virus are endemic in Irish prisons. A third of prison entrants were committed to prison for the first time. Only a small number of first time entrants were infected with one or more of the viruses. These findings confirm the need for increased infection control and harm reduction measures in Irish prisons.

Decrease in circulating tumor cells as an early marker of therapy effectiveness.

As melanoma cells are present in the circulation of many patients with this cancer, decreases in their number could provide an early indication of therapy effectiveness. To evaluate this possibility, we examined the effect of treatment with a melanoma vaccine on the number of melanoma cells present in the circulation. PCR was used to detect melanoma cells that expressed the melanoma-associated antigens MART-1, MAGE-3, tyrosinase and/or gp100 in 91 patients with melanoma. Melanoma cells that expressed one or more of these markers were present more often in advanced disease, i.e. in 80% of patients with advanced stage IV compared to in less than one-third of patients with less advanced disease. We then measured circulating melanoma cells in a subset of 43 of these patients who were treated with a polyvalent, shed antigen, melanoma vaccine. The vaccine contains multiple melanoma-associated antigens including MART-1, MAGE-3, tyrosinase and gp100. Immunizations were given intradermally q2-3 weeks x4 and then monthly x3. Prior to vaccine treatment, circulating melanoma cells were detected in 14 (32%) patients. Following 4 and 7 months of vaccine treatment, melanoma cells that expressed any of these markers were present in only nine (21%) and three (7%) of patients, respectively. Thus, vaccine therapy was associated with clearance of melanoma cells from the circulation in 78% of initially positive patients. As the number of these cells declined steadily with increasing length of therapy, it is unlikely that this was due to a random change in their number. Rather it suggests that the decline was a result of the therapy. These observations suggest that the presence of melanoma cells in the circulation is related to the extent of the melanoma, and that their disappearance may provide an early marker of the efficacy of therapy. However, the practical utility of assaying circulating tumor cells as a guide to the effectiveness of therapy or of prognosis will need to be confirmed by correlations with clinical outcome.

Identification of HLA-A*03, A*11 and B*07-restricted melanoma-associated peptides that are immunogenic in vivo by vaccine-induced immune response (VIIR) analysis.

With the discovery of increasing numbers of tumor antigens, there is a need to rapidly determine whether these antigens and the individual peptides they express are able to stimulate immune responses in vivo and thus, can be used to construct cancer vaccines. In this study we used the method of vaccine-induced immune response (VIIR) analysis to identify multiple immunogenic peptide epitopes derived from several melanoma associated antigens and presented by HLA-A*03, A*11 and B*07. Thirty-one patients with melanoma were immunized to a polyvalent vaccine containing multiple antigens, including MAGE-3, Melan A/MART-1, gp100 and tyrosinase. Their peripheral blood was tested for peptide-specific, vaccine-induced CD8+ T cell responses before and after immunization using an enzyme-linked immune spot (ELISPOT) assay with panels of peptides restricted by these three alleles. The peptides were selected for immunogenic potential based on their strong binding affinity in vitro to HLA-A*03, A*11 or B*07. Overall, 60% of the 20 peptides studied were recognized by at least one patient and 50% of the patients showed a vaccine-induced CD8+ T cell response to at least one peptide that matched their HLA specificity. We conclude that VIIR analysis is an effective strategy to directly identify immunogenic peptides that are good candidates for vaccine construction.

HLA-independent heterogeneity of CD8+ T cell responses to MAGE-3, Melan-A/MART-1, gp100, tyrosinase, MC1R, and TRP-2 in vaccine-treated melanoma patients.

An important element in melanoma vaccine construction is to identify peptides from melanoma-associated Ags that have immunogenic potential in humans and are recognized by CD8+ T cells in vivo. To identify such peptides, we evaluated HLA-A*02+ melanoma patients immunized to a polyvalent vaccine containing multiple Ags, including MAGE-3, Melan-A/MART-1, gp100, tyrosinase, melanocortin receptor (MC1R), and dopachrome tautomerase (TRP-2). Using a filter spot assay, we measured peripheral blood CD8+ T cell responses, before and after immunization, to a panel of 45 HLA-A*0201-restricted peptides derived from these Ags. The peptides were selected for immunogenic potential based on their strong binding affinity in vitro to HLA-A*0201. Vaccine treatment induced peptide-specific CD8+ T cell responses to 22 (47.8%) of the peptides. The most striking finding was the HLA-independent heterogeneity of responses to both peptides and Ags. All responding patients reacted to different combination of peptides and Ags even though the responding patients were all A*0201+ and the peptides were all A*0201-restricted. From 9 to 27% of patients developed a CD8+ T cell response to at least one peptide from each Ag, but no more than 3 (14%) reacted to the same peptide from the same Ag. This heterogeneity of responses to individual peptides and Ags in patients with the same haplotype points to the need to construct vaccines of multiple peptides or Ags to maximize the proportion of responding patients.

Stimulation of CD8+ T cell responses to MAGE-3 and Melan A/MART-1 by immunization to a polyvalent melanoma vaccine.

A critical requirement for cancer vaccines is that they stimulate CD8+ T cell responses. In this study, we tested the ability of a polyvalent melanoma vaccine to induce CD8+ T cell responses to the melanoma associated antigens MAGE-3 and Melan A/MART-1. Fifteen HLA-A2+ patients with resected malignant melanoma were immunized with the vaccine s.c. every 2-3 weeks. CD8+ T cells in peripheral blood reacting to HLA-A2 restricted epitopes on MAGE-3 (FLWGPRALV) and Melan A/MART-1/(AAGIGILTV) were quantitated using a filter spot assay at baseline and following 4 immunizations. Vaccine immunization induced CD8+ T cells reacting to one or both of these peptides in 9 of the 15 (60%) patients. These cells were CD8+ and HLA-A2 restricted, as reactivity was abrogated by monoclonal antibodies (MAbs) to CD8 and class I HLA, but not by anti-CD4. All responding patients remained recurrence-free for at least 12 months (median 15 months, range 12 to >21 months), whereas melanoma recurred within 3-5 months in non-responders. The differences in outcome were unrelated to differences in disease severity or overall immunological competence between responders and non-responders. Our results demonstrate directly that MAGE-3 and Melan A/MART-1 can stimulate CD8+ T cell responses in humans, and suggest that these responses are protective and surrogate markers of vaccine efficacy.

Detection of antibodies to melanocytes in vitiligo by western immunoblotting.

To more fully define the nature of the antibody response to melanocytes which is associated with vitiligo, a Western immunoblot assay was used to test the sera of 28 patients with vitiligo (21 with active non-segmental, and 7 with stable segmental diseases) and 26 normal individuals for antibodies to antigens in detergent extracts of melanocyte membrane fractions. Antibodies to melanocytes were found in 26 (93%) of the patients with vitiligo, and in 16 (62%) of the control individuals. Patients with vitiligo and control individuals both had antibodies to an 80 approximately 83 kD antigen. The patient with vitiligo, in addition, had antibody responses to antigens with MWs of 45, 65, and 110 kD. Antibodies to these antigens were present in 46, 25, and 31% of vitiligo patients, but in only 19%. 0%, amd 0%, respectively, of the normal individuals. The heterogeneity of the antibody responses to melanocytes in vitiligo was further confirmed by the presence of antibodies to at least 3 distinct antigens in one-third of vitiligo patients but in none of the normal individuals. There was no difference in antibody response between patients with generalized and segmental vitiligo, suggesting that the pathogenesis of diseases was similar in both cases.

Schistosoma mansoni hexokinase: cDNA cloning and immunogenicity studies.

DNA encoding a Schistosoma mansoni hexokinase (SHEX) was amplified from cDNA by the polymerase chain reaction using opposing oligonucleotide primers designed to hybridize with two short segments of hexokinase coding sequences that are well-conserved through evolution. The resulting DNA fragment was then used as a probe to identify a full-length hexokinase cDNA clone. SHEX cDNA encodes a 50-kDa protein that is approximately 46% homologous to rat hexokinase, 40% to rat glucokinase, and 34% to yeast hexokinase A. SHEX coding DNA was expressed within Escherichia coli cells and the 50-kDa recombinant product (rSHEX) was partially purified. Mice repeatedly immunized with rSHEX produced antibodies which recognize rSHEX but this offered no significant protection against subsequent cercarial challenge. On Western blots, rSHEX is weakly recognized by antisera against rat brain hexokinase but not by sera from three strains of mice experimentally infected with S. mansoni parasites or from numerous human schistosomiasis patients. Thus, unlike other reported S. mansoni glycolytic enzymes, hexokinase appears to be poorly immunogenic during schistosome infection and of limited potential as a vaccine candidate.

T and B epitope determination and analysis of multiple antigenic peptides for the Schistosoma mansoni experimental vaccine triose-phosphate isomerase.

Schistosoma mansoni triose-phosphate isomerase (TPI), a glycolytic enzyme, was originally identified as the target of the mAb M.1 that conferred protection when the antibody was administered in vivo. In this study we increase the evidence that schistosome TPI is a potential vaccine Ag by showing that it also a potent inducer of IL-2 and IFN-gamma production (Th1 responses), driving production of these cytokines in the same cell populations of infected animals that have high Th2 responses directed at other parasite egg Ag. With the goal of synthetic peptide vaccine design, rTPI was used to determine specific T and B cell epitopes recognized by two strains of mice representing high and moderate responders (C57Bl/6J and CBA/J). All selected epitopes were from nonconserved regions of TPI and thus parasite-specific. We then defined minimal size immunoreactive epitopes and synthesized four-armed multiple antigenic peptides (MAP) consisting of T and B cell epitopes that could be recognized by both strains of mice in the same molecule. Characterization of the immunoreactivity of the MAP showed that higher antibody recognition of the MAP was attained when the B cell epitope was placed on the amino termini relative to the T cell epitope, whereas equivalent immunoreactivity occurred for the T cell epitopes when located at either position. Most interesting was the finding that one of the minimal T cell epitopes, when incorporated into the MAP, required enlarging to retain immunoreactivity. Finally we showed that both the full-length TPI molecule and the final version of the MAP were immunogenic to T cells in naive animals and induced cross-recognition in the form of IL-2 and IFN-gamma production.

Candidate vaccine antigens that stimulate the cellular immune response of mice vaccinated with irradiated cercariae of Schistosoma mansoni.

Vaccination with radiation-attenuated cercariae confers the highest levels of resistance to challenge infection in experimental schistosomiasis and requires Ag-specific T cells. Therefore, this study aimed to identify specific Ag that stimulate the cellular immune response of mice vaccinated with irradiated cercariae of Schistosoma mansoni. Four experimental groups representing different levels of resistance in the vaccine model (C57BL/6J versus CBA/J mice vaccinated with 15- or 50-krad irradiated cercariae) were compared for in vitro lymphocyte proliferation and lymphokine production. Adult worm extracts fractionated by isoelectric focusing were used as Ag. Lymphocyte proliferation of all groups was limited to three consecutive isoelectric fractions (pH 4.6-6.3). Interestingly, the antibody response of these mice was directed to Ag in the same isoelectric fractions, three of which had previously been identified as paramyosin, heat shock protein 70, and the integral membrane protein Sm23. These Ag as well as two 28 kDa proteins, triosephosphate isomerase and glutathione S-transferase, in purified native or recombinant form or as a synthetic peptide, stimulated lymphocyte proliferation. Lymphocytes of vaccinated C57BL/6J mice generally showed higher levels of proliferation than did CBA/J mice. Interestingly, cells of once-vaccinated mice responded better than did cells of mice vaccinated three times. Lymphokine assays demonstrated that IL-2 and IL-4 was generally reduced after multiple vaccinations and varied qualitatively as well as quantitatively between mouse strains. This study substantiates that the five Ag, paramyosin, heat shock protein 70, triosephosphate isomerase, glutathione S-transferase, and the integral membrane protein Sm23, are important candidates for a defined antischistosomal vaccine.

T and B cell epitope mapping of SM23, an integral membrane protein of Schistosoma mansoni.

SM23 is an integral membrane protein of the blood-vessel dwelling parasitic worm Schistosoma mansoni. This protein has been detected with antibodies in all stages of the parasite found in the human host, notably the lung stage, and therefore is of interest as a vaccine candidate. In addition SM23 has been shown to be a member of a proposed new superfamily of membrane proteins whose structures do not conform to the previously known classifications. To date there are 13 members including ME491 (CD63, Pltgp40), CD9 (p23), TAPA-1, CD37, CD53, MRC OX-44, CO-029, MRP-1, L6, the gene product of TI-1, the target of mAb AD-1, SM23, and SJ23 (the Schistosoma japonicum homologue). Most of these molecules except for those in the two blood vessel-dwelling parasites are found in membranes of hemopoietic and/or malignant cells and all have unknown function. In this study we used recombinantly expressed full-length and partial molecules as well as synthesized peptides to map T cell and B cell epitopes of SM23. The two predicted external hydrophilic domains were found to be highly immunogenic and contained several B cell epitopes. There were at least four T cell epitopes in the large hydrophilic domain. One segment of 23 amino acids contained both a T cell and B cell epitope as well as the putative glycosylation site. This particular segment was recognized by immune sera and cells of every mouse strain tested. The elucidation of these epitopes demonstrates the immunogenic nature of this molecule and raises questions as to the role of SM23 in the host/parasite relationship.

Comparison of irradiated-cercaria schistosome vaccine models that use 15- and 50-kilorad doses: the 15-kilorad dose gives greater protection, smaller liver sizes, and higher gamma interferon levels after challenge.

The protection and immune response to infection caused by the parasite Schistosoma mansoni were studied by comparison of two murine irradiated-vaccine models. Mice were exposed from 1 to 4 times to infective-stage cercariae attenuated with either a moderate dose (15 kilorads) or a high dose (50 kilorads) of radiation. Seven weeks after challenge infection, the mice were assessed for resistance, liver size, and lymphokine responses to parasite antigens. Both vaccine models showed high levels of protection, but the moderate-dose model proved superior in that mice in those groups achieved higher levels of resistance in fewer exposures. Additionally, the mice exposed three times and four times to moderately irradiated cercariae all had significantly lower liver weights independent of worm burden. Assessment of lymphokine production by the spleen cells at the time of perfusion showed that gamma interferon was the only lymphokine of those measured that was differentially produced in the two models and correlated with a decrease in size of in vitro granulomas. The findings suggest that a selected vaccine regimen may lead to greater resistance and decreased liver pathology, the latter of which appears to be mediated by induction of gamma interferon.

T cell clones for antigen selection and lymphokine production in murine Schistosomiasis mansoni.

The role of T cells in immunity to murine schistosomiasis was examined through the use of T cell clones that recognize the live schistosomulum stage of Schistosoma mansoni. T cell clones of three different phenotypes were isolated and expanded into long term culture from lymph nodes of C57B1/6J mice vaccinated with irradiated S. mansoni larvae. They were characterized by surface markers, lymphokine production, and functional assays. The m.w. range of the Ag recognized by one clone was identified through nitrocellulose blotting and confirmed with a preparation of the putative protein made by immunoaffinity purification. All but one of the clones were CD4+, CD5+, Th cells. One clone, 35, produced Il-2 and IFN-gamma and was designated a TH1 clone. The others were designated TH2 clones because of Il-4 production. One clone was CD8+ and failed to show help. Clone 35 recognized live schistosomula and produced Il-2 when presented a 27-kDa protein from nitrocellulose. It proliferated in response to purified Ag. Clone 35 participated along with macrophages to induce up to 98% killing of live schistosomula in vitro. IFN-gamma and TNF-alpha were essential to the killing mechanism whereas Il-1, Il-2, and Il-4 were not required. This study has approached Ag identification for vaccine development from the point of view of T cells and showed that TH1 cells are essential to in vitro macrophage killing of schistosomula in murine schistosomiasis.

Intravenous beta agonist in severe acute asthma.

STUDY OBJECTIVE: To determine whether salbutamol is more effective in treating severe asthma when given intravenously or by inhalation. DESIGN: Randomised trial of short term response to intravenous versus nebulised salbutamol in acute severe asthma. SETTING: District general hospital (secondary care centre). PARTICIPANTS: 76 patients aged 16-70 admitted to hospital with acute severe asthma (peak expiratory flow rate less than 50% of predicted) during study period. Five withdrawn because of adverse effects of treatment or non-response. Of remaining 71, 34 allocated to nebuliser group and 37 to intravenous treatment group. Patients with history of cardiovascular disease or recent corticosteroid or intravenous bronchodilator treatment excluded. Admission characteristics similar in the two groups. INTERVENTIONS: All patients given 5 mg nebulised salbutamol on admission before randomisation plus 200 mg hydrocortisone bolus intravenously and 35% inspired oxygen throughout. Nebuliser group received two more 5 mg doses of nebuliser salbutamol at 30 minutes and 2 hours; intravenous group received 4 hours' continuous salbutamol infusion (12 micrograms/min) starting at 30 minutes plus supplementary intravenous potassium chloride. No other bronchodilators used. ENDPOINT: Change in peak expiratory flow rate over 4 hours. MEASUREMENTS AND MAIN RESULTS: Peak expiratory flow rate improved more in intravenous group (25.2%) than in nebuliser group (14.3%) (p less than 0.01, 95% confidence interval 2.4 to 19.1%). Tachycardia caused two withdrawals from intravenous group; non-response caused three withdrawals from nebuliser group. CONCLUSIONS: Intravenous salbutamol is more effective than nebulised salbutamol in acute severe asthma but may have unacceptable cardiovascular effects.

Mechanisms of placentofetal blood flow.

The average length of the umbilical cord at delivery is 21 +/- 1 in., but lengths vary from 10 to 50 in. When distended and carrying blood between the fetus and the placenta, the cord is longer; the distended areteries and vein are spiraled about each other. No physiologic data explain the manner by which blood flows from the placenta to fetus. When pressures (60-80 mmHg in arteries 10-20 mmHg in the vein) are recorded simultaneously, arterial pulse pressure has been observed to rise and umbilical vein pressure to fall. They are 180 degrees out of phase. When necessary physical factors are taken into account, it is found that the system satisfies all requirements of a pump known as one of the pulsometer type. This system is one that is widely used by engineers, but it is unique among physiologic systems.

Hydatidiform mole: a vascular congenital anomaly.

The thesis is advanced that a critical time for development of embryonic blood vessels in the placenta is 13 to 21 days after conception, especially during days 18 to 21. Dietary requirements at this time are specific and demanding for nutritional precursors of thymidine which is an important constituent of DNA. Folic acid and histidine are specifically essential for thymidine synthesis. These are reviewed with respect to cultures and socioeconomon levels in societies where occurrence of hydatidiform mole is endemic. Specific nutritional deficiencies are discussed in relation to kinds of diets and ways of food preparation. When specific dietary requirements are lacking at a time of high need, embryo death, abnormality, and/or avascularity of trophoblastic placental villi may be the earliest pathogenic signs of hydatidiform mole.