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Objectives: To investigate whether immunization with recombinant HIV-1 envelope protein derived from a clinical isolate could protect macaques from infection with an in vivo passaged chimeric simian-human immunodeficiency virus (SHIV)., Design and Methods: A total of 16 animals were studied from which three groups of four animals were immunized with vaccine formulations of the CC-chemokine receptor-5-binding recombinant gp120 of HIV-1W6.1D. Four weeks after the last immunization, all 16 animals were intravenously challenged with in vivo passaged SHIV derived from the same HIV-1 group B clinical isolate (W6.1D) as the vaccines., Results: Vaccine protection from infection was demonstrated in 10 out of 12 macaques immunized with recombinant gp120. Complete protection from infection was achieved with all of the animals that received the SBAS2-W6.1D formulation, a potent inducer of both T-cell and humoral immune responses. Partial protection was achieved with SBAS1-W6.1D, a formulation based on immunomodulators known to induce T-cell responses in humans. In vaccinated animals that were infected, virus load was reduced and infection was delayed., Conclusions: In a relatively large number of primates, vaccine efficacy was demonstrated with a clinically relevant HIV-1 vaccine. These results reveal that it is possible to induce sterilizing immunity sufficient to protect from infection with SHIV which was passaged multiple times in vivo. Our findings have implications for current HIV-1 clinical vaccine trials and ongoing efforts to develop safe prophylactic AIDS vaccines.

The use of HIV-1 env/SIVmac chimeric viruses expressing divergent HIV-1 envelopes of clinical isolates, facilitates homologous and heterologous evaluation of various recombinant HIV-1 envelope vaccine candidates in lower primates. In this study we compare the in vitro and in vivo infectivity, via intravenous (IV) and intravaginal (IVAG) routes of infection, of stocks of chimeric viruses expressing env from four different clade B HIV-1 isolates. The TCID50/ml was 7.1 x 10(4), 1.0 x 10(4), 6.3 x 10(4), and 1.2 x 10(3) for SHIVsf13, SHIVHan2, SHIVNM-3rn, and SHIVW6.1D, respectively, with a MID50/ml upon IV inoculation of 3.2 x 10(3), 3.2 x 10(4), 3.2 x 10(4), and 3.2 x 10(3), respectively. The same SHIVsf13 stock was infectious after IVAG administration, requiring a 300-fold higher virus dose. Plasma antigenemia and cell-associated viremia were generally highest at weeks 2 or 4 after infection and decreased to subdetectable levels after 8-12 weeks. All infected animals tested developed anti-HIV-1 gp120 antibodies. Inoculated virus dose showed no (linear) quantitative correlation with cellular virus load, duration of viremia, plasma antigenemia, and anti-gp120 antibody titers. No significant changes in peripheral blood CD4 cell levels were observed and none of the animals has shown evidence of disease progression to date (i.e., 13 months postinfection). Four in vivo passages of cell-associated SHIVW6.1D did not result in increased virulence. Vaccine development studies in macaques monkeys have become feasible with the use of various clade B HIV-1 env SHIV chimeras., (Copyright 1997 Academic Press.)

Inoculation of macaques with live attenuated SIV strains has been shown to protect against subsequent challenge with wild-type SIV. The protective mechanism(s) remain obscure. To study the effect in more detail, we have investigated the role of virus-specific CTL responses in macaques infected with an attenuated SIV strain (pC8), which has a four-amino acid deletion in the nef gene, as compared with the wild-type SIVmac32H clone (pJ5). Cynomolgus macaques infected with pC8 were protected against subsequent challenge with pJ5 and did not develop any AIDS-like symptoms in the 12 months after infection. The pC8-induced protection was associated with high levels of virus-specific CTL responses to a variety of viral antigens. In contrast, pJ5-infected macaques had little, if any, detectable CTL response to the viral proteins after three months. The latter group of macaques also showed increased Fas expression and apoptotic cell death in both the CD4(+) and CD8(+) populations. In vitro, pJ5 but not pC8 leads to an increase in FasL expression on infected cells. Thus the expression of FasL may protect infected cells from CTL attack, killing viral-specific CTLs in the process, and providing a route for escaping the immune response, leading to the increased pathogenicity of pJ5. pC8, on the other hand does not induce FasL expression, allowing the development of a protective CTL response. Furthermore, interruption of the Fas-FasL interaction allows the regeneration of viral-specific CTL responses in pJ5-infected animals. This observation suggests an additional therapeutic approach to the treatment of AIDS.

The effect of hepatic nerve stimulation and norepinephrine (NE) on the laser Doppler signal from the surface of the perfused rat liver was tested. The livers from male Wistar rats were perfused in situ via the portal vein with Krebs-Henseleit buffer containing 20% bovine erythrocytes (37 degrees C, pH 7.4) and total liver blood flow (TLBF) was by timed collection of effluent. Portal vascular resistance (PVR) was calculated from the pressure difference across the liver. Linearity of laser Doppler flowmetry (LDF) with TLBF was confirmed in all preparations. Stimulation of the hepatic nerves (2 ms, 20 V) was performed at frequencies between 0.5 and 20 Hz (n = 11). NE was added to the buffer at concentrations between 10(-10) and 10(-6) M (n = 8). A stimulus-dependent rise in PVR occurred during hepatic nerve stimulation (basal, 3.11 +/- 0.26 dyn s cm-5) and NE administration (basal, 2.62 +/- 0.29 dyn s cm-5), with a maximum effect at 20 Hz (311 +/- 45%) and 10(-6) M (591 +/- 72%), respectively. Both LDF and TLBF fell during nerve stimulation and NE. A linear relationship (r = 0.99; p < 0.001) between change in TLBF (%) and LDF flux (%) was found for NE (10(-10) to 10(-6) M). During nerve stimulation, the fall in TLBF and LDF flux was linear with the logarithm of stimulus frequency and reached a maximum at 10 and 20 Hz, respectively. At a stimulus frequency of 20 Hz, the change in LDF was significantly different from the change in TLBF (p < 0.001). We conclude from our findings that during high-frequency hepatic nerve stimulation, LDF underestimates TLBF.

Two cynomolgus macaques were infected with a genetically complex challenge stock of simian immunodeficiency virus (SIVmac251-32H). One animal developed SIV-induced disease and was sacrificed at 16 months postinfection. The second remained healthy until it too was sacrificed at 20 months postinfection. The polymerase chain reaction (PCR) was used to amplify env gp120-coding sequences from provirus present in samples of blood, spleen, and inguinal lymph node taken from both animals on the day of sacrifice. The proviral burden present in each of the tissue samples was also determined using a quantitative PCR assay. The proviral burdens in the blood, spleen, and inguinal lymph node of the healthy animal (I225) were similar. This was not the case for animal I227, in which the burden in the inguinal lymph node was much higher than for blood or spleen. Phenogram analysis of the hypervariable V1 region of env revealed that the diversity of nucleotide sequences recovered from each tissue of both macaques were similar and overlapping. Some selected amino acid differences were observed that were specific for a tissue or one of the macaques. However, the results do not suggest that the overall evolution of env in provirus populations recovered from lymphoid tissues is distinct from that recovered from the blood.

In order to develop a successful subunit vaccine against infection with the human immunodeficiency virus (HIV), protective immune effector functions must be identified. Until now, there has been only indirect evidence that HIV-specific cytotoxic T lymphocytes (CTLs) fulfill this role. Using the macaque simian immunodeficiency virus (SIV) model, the protective potential of nef-specific CTLs, stimulated by vaccination, was examined in animals challenged with a high intravenous dose of the pathogenic simian immunodeficiency virus, SIVmac251(32H)(pJ5). An inverse correlation was found between the vaccine-induced nef-specific CTL precursor frequency and virus load measured after challenge. In addition, the early decline in viraemia, observed in both vaccinated and unvaccinated control animals was associated with the development of virus-specific CTL activity and not with the presence of virus-specific neutralizing antibodies. The results imply that vaccines that stimulate strong CTL responses could protect against HIV infection.

Objective: To evaluate the efficacy of immunopurified class I human histocompatibility leukocyte antigen (HLA) to protect against SIV infection., Methods: HLA class I antigens were immunopurified from a human B-lymphoblastoid cell line. Groups of four macaques were vaccinated subcutaneously with four doses of the immunogen in adjuvant, or with adjuvant alone and subsequently challenged intravenously with 10 median monkey infectious doses of cell-free SIVmac-32H. Infection was determined by polymerase chain reaction for SIVmac proviral DNA and by virus isolation. Antigen-specific humoral and cellular immune responses were monitored., Results: Macaques immunized with the HLA molecules produced anti-HLA class I antibodies that inhibited SIV replication in vitro and downregulated autologous T-cell proliferation against irradiated C8166 cells. They were partially protected (two out of four) from virus infection for at least 33 weeks when challenged with SIV grown in human cells. All four control animals were infected., Conclusions: This demonstration of partial protection, together with our previous work reporting that vaccination with allogenic cynomolgus lymphocytes can protect against challenge infection with SIV grown in simian cells, suggests that allogenic immune response induced before or during establishment of HIV infection may have important implications for AIDS disease progression.

Polymerase chain reaction based assays, which amplify a region of the gag gene, have been developed for the direct detection of simian immunodeficiency virus (SIV) DNA sequences in the blood of experimentally infected cynomolgus macaques. In macaques infected with a characterised virus pool (11/88 pool SIVmac 32H), an assay employing a single round of amplification was found to be highly sensitive and specific. However, in animals infected with the SIV molecular clones J5 and C8 (Rud et al., J. Gen. Virol. 75, 529-543), it was necessary to use two rounds of amplification and nested primer pairs in order to achieve sensitivity > 90%. In order to differentiate macaques infected with either of the two genetically distinct SIV clones, J5 or C8, a third PCR based assay has been developed, which amplifies a 492 bp region of the nef gene. Sequence differences between the nef genes of the two molecular clones enabled the PCR product amplified from each virus to be distinguished by restriction analysis. These sensitive and specific assays complement virological detection of SIV and enable superinfection studies to be evaluated; a prerequisite for the testing of live attenuated immunodeficiency virus vaccines.

Sequences of the simian immunodeficiency virus (SIV) rev mRNA transcripts were characterized from peripheral blood lymphocytes (PBLs) of two macaques experimentally infected with SIVmac251 (32H reisolate). This analysis has demonstrated that the complex splicing patterns observed for the rev mRNA transcripts originally identified in vitro is not an artifact of tissue culture, but is also found in vivo.

Objective: The etiology of rheumatoid arthritis (RA) is unknown, and the possibility that an infectious agent is involved has not been excluded. Lentiviruses can cause chronic arthritis in humans and in animals and have been suggested as candidate agents in RA. We therefore tested for the presence of lentiviruses and also for human T cell leukemia virus type I (HTLV-I)/HTLV-II in cells from patients with RA., Methods: We used the polymerase chain reaction with degenerate primers designed to recognize highly conserved nucleotide sequences from 5 different pathogenic lentiviruses. This method allowed the detection of at least 1 infected cell/20,000 uninfected cells in control experiments., Results: Testing of synovial cells and blood cells from patients with early RA and patients with established RA did not yield any specific viral product., Conclusion: Our results do not support the presence of lentiviruses or HTLV-like sequences in RA.

It has been proposed that the laser Doppler flow (LDF) signal from the surface of the rat liver is almost exclusively a measure of hepatic arterial and not of total liver blood flow and therefore that LDF is not a suitable technique for the measurement of blood flow in the hepatic microcirculation. The objective of the present study was twofold: (i) to establish that liver blood flow is homogeneously distributed and (ii) to assess the behavior of the LDF signal during changes in hepatic perfusion. When 51Cr-labeled microspheres were injected into the portal vein (n = 12), no significant differences in the relative flow (cpm/lobe to cpm/liver) to each of the liver lobes were found nor was there any difference in the ratio of flow to the outer 1-2 mm of lobe as compared to that to the "core" of the liver. Temporary occlusion of the hepatic artery and the portal vein caused approximately 13% (n = 7, P < 0.001) and approximately 74% (n = 7, P < 0.001) fall in LDF signal, respectively. Diversion of flow from the anterior to the posterior lobes (n = 5) caused a 97.9 +/- 21.1% (SD, P < 0.001) rise in LDF signal in the posterior lobes. Zero-flow LDF signal was found to represent 13.0 +/- 4.1% of maximum. Hemorrhage (in 1.5-ml aliquots) was associated with a fall in mean arterial pressure (MAP) and LDF signals. A linear relationship between MAP and the LDF signal (r > 0.9) was found. Reinfusion of blood caused both MAP and the LDF signal to return to normal. We conclude that (i) blood flow in rat liver is homogeneously distributed; (ii) the LDF signal from the liver surface responds in a manner predicted by conventional theories of hepatic hemodynamics during alteration, either independent or combined, in hepatic arterial and portal venous blood flow; and (iii) LDF may be used to measure relative changes in hepatic perfusion but problems associated with zero-flow signal and intersite variability preclude its quantification in absolute flow units.

Three cynomolgus macaques were immunized with recombinant envelope protein preparations derived from simian immunodeficiency virus (SIV). Although humoral and cellular responses were elicited by the immunization regime, all macaques became infected upon challenge with 10 MID50 of the 11/88 virus challenge stock of SIVmac251-32H. The polymerase chain reaction was used to amplify proviral SIV gp120 sequences present in the blood of both immunized and control macaques at 2 months post-infection. A comparison of the predominant sequences found in the region from V2 to V5 of gp120 failed to differentiate provirus recovered from either immunized or control animals. A detailed investigation of sequences obtained from the hypervariable V1 region identified a mixture of sequences in both immunized and control macaques. Some sequences were identical to those previously detected in the virus challenge stock, whereas others had not been detected previously. Phenogram analysis of the new V1 sequences found in immunized animals revealed that they were quite distinct from those from the virus challenge stock and that they included alterations to potential N-linked glycosylation sites. In contrast, new sequence variants recovered from the control animals were closely related to sequences from the virus challenge stock. The difference in diversity of new V1 sequences recovered from immunized and control macaques was highly significant (P < 0.001). Thus, the presence of pre-existing immune responses to SIV envelope protein is associated with greater genetic change in the V1 region of gp120. These data are discussed in relation to the epitopes of SIV gp120 that may confer protection from in vivo challenge.

Two cynomolgus macaques were infected with a genetically complex challenge stock of simian immunodeficiency virus (SIVmac251-32H). The polymerase chain reaction (PCR) was used to amplify the env gp41, rev, and nef overlapping coding sequences from provirus present in the blood of both animals at 1, 6, and 15 months post infection (p.i.). The predominant, env sequences found in both animals at the three time points were very similar to that found in the original 11/88 challenge stock. The functionally important hydrophobic fusion and membrane-spanning domains within gp41 remained conserved throughout the course of infection. Nucleotide variation within the region corresponding to the REV response element (RRE) was limited to four positions, none of which were predicted to cause any significant disruption to the secondary structure of the RRE. Very little genetic variation was observed in and around the cluster of potential glycosylation sites of the external portion of gp41. However, the existence of a previously assigned variable region elsewhere in the cytoplasmic domain of gp41 was confirmed. The three gene loci (env, rev, and nef) examined varied independently. All changes in the predominant protein sequences were brought about by single nucleotide substitutions only. After 15 months of infection with SIV, 1 animal was sick from SIV-induced disease whereas the other remained healthy. In-frame stop codons within the transmembrane protein occurred with a much greater frequency in the healthy animal.

Two cynomolgus macaques were infected with a complex, but characterized, challenge stock of simian immunodeficiency virus (SIVmac251 32H). The polymerase chain reaction was applied in a temporal sequence analysis to determine the sequences of the gp120 region of the SIV env gene, which were present in the blood of both macaques at 1, 6, and 15 months postinfection (p.i.). At 1 month p.i. selected sequences, which had been present in the original virus challenge stock, were reisolated. At later times, new sequences emerged, which had not been detected in the original virus challenge stock. Changes in sequence were restricted to specific regions of gp120, notably those equivalent to V1, V2, V4, and V5 of HIV-1, but not V3. The diversity and the rate of appearance of new sequences in the V1 region suggest that genetic evolution occurs by mechanisms in addition to nucleotide substitutions. These results are discussed in relation to the role of the envelope protein in the generation of protective immunity against infection with immunodeficiency viruses.

The polymerase chain reaction (PCR) is often used to assess the diversity of viral nucleotide sequences present in various biological samples (e.g., blood and tissues). However, it is not clear how reproducible this approach may be. DNA was extracted from the peripheral blood lymphocytes of a macaque that had been infected, experimentally, with SIVmac251-32H 6 months previously. The nef gene was then amplified by the PCR on three separate occasions from this same template preparation. A panel of clones was prepared from the product of each PCR, the entire nef gene was sequenced and the sequences obtained compared with each other. Phenogram analysis revealed that within each panel the same degree of sequence diversity was observed between clones. Furthermore, when the sequences obtained from all three panels were compared, the overall sequence diversity observed was no greater than that observed for each panel individually. These data indicate that the analysis of sequence diversity in SIV 'quasi-species' populations by the PCR is reliable and, more important, reproducible.

Little is known about the performance of the laser Doppler flowmeter (LDF) with changes in flow characteristics in the hepatic microcirculation. Red blood cell (RBC) flux, as measured by LDF, is sensitive to alterations in RBC velocity and RBC concentration, and both parameters must be considered when evaluating the technique. In vitro, linearity of LDF signal with RBC velocity up to 4 mm/s (r greater than 0.99) and RBC volume fractions up to 1.5% were demonstrated (r greater than 0.98). At higher velocities and volume fractions, RBC flux was underestimated. In portally perfused rat liver, LDF output was linearly related to total liver blood flow (TLBF; r greater than 0.9) for perfusate hematocrits between 2.5 and 40%, although the slope varied between preparations. At constant TLBF (2 ml.min-1.g-1), the LDF output changed linearly with perfusate hematocrit up to 20% (r greater than 0.99) but underestimated the increase at 40%. These results suggest that the LDF responds linearly to velocity-mediated flow changes but that it may underestimate a change if mediated through alteration in tissue RBC concentration. With hepatic nerve stimulation, TLBF fell to 51 +/- 14% of prestimulation (P less than 0.001), whereas the LDF output and superficial flow measured by 85Kr clearance fell to 12 +/- 10 and 14 +/- 10% (both P less than 0.01 vs. TLBF), respectively, suggesting that the LDF may be used to follow rapid flow changes in the periphery of the liver.

The virus structural genes gag and env (both gp120 and gp41 regions) of the 32H isolate of SIVmac251 were amplified using the polymerase chain reaction (PCR). The proviral template used in the PCR was DNA isolated from cells used to prepare several experimental SIV vaccines, which have been tested in simians, and a standard challenge stock of virus, which has been used in international collaborative studies. The PCR products were cloned and the nucleotide sequences of several clones were determined for each gene. From a comparison of the sequences obtained the predominant amino acid sequences of gag and env were predicted and the degree of sequence heterogeneity was determined. Conserved and more variable regions of each protein were identified. The gp120 region of env was more heterogeneous than gag or the transmembrane protein of env (gp41). Within gp120, sequence variability was concentrated to specific regions equivalent to the V1, V2, and, to a lesser extent, the C1 regions identified for human immunodeficiency virus type 1 (HIV-1). In contrast the region equivalent to the hypervariable "V3-loop" of HIV-1 was highly conserved. The implications of the data is discussed in relation to the ability of this virus stock to prepare effective vaccines against SIV.

Intradermal injections of normal saline and different concentrations of bradykinin were made into the forearms of healthy volunteers. Cutaneous blood flow was recorded just outside and over the centre of the weals by laser Doppler flowmetry (LDF), and flare area and weal volume were measured. There were concentration-related changes in the mean LDF output adjacent to the weal, flare area and weal volume. LDF recordings performed over the centre of the weals, however, failed to show any dose-response relationship. When similar concentrations of bradykinin were injected intradermally and the skin response measured by the above parameters, there was less variation in the mean LDF output adjacent to the weal than flare or weal measurements. The non-invasive technique of LDF is a useful, objective and sensitive technique of quantifying the skin blood flow changes induced by intradermal bradykinin and provides an alternative method of quantifying skin response to intradermal bradykinin to measurement of flare or weal sizes.

The L1 gene of human papillomavirus type 16 (HPV-16) driven by the vaccinia virus major late 4b gene promoter has been inserted into three different sites of the vaccinia virus genome. Insertion into the thymidine kinase (TK) gene was achieved by selection of TK- mutants in BUdR on TK- cells. Insertion into two vaccinia virus serine protease inhibitor (serpin) genes was achieved by co-insertion of the Escherichia coli xanthine guanine phosphoribosyltransferase gene linked to the vaccinia virus 7.5K promoter and selection of mycophenolic acid-resistant recombinant viruses. Each recombinant virus expressed a 57K L1 protein at similar levels and with similar kinetics. However, immunization of mice with these recombinant viruses induced different levels of antibody to the L1 protein. Viruses lacking serpin genes B13R and B24R induced significantly higher antibody levels than did viruses lacking the TK gene. The presence of functional B13R and B24R gene products is therefore somehow immunosuppressive at least for antibody responses to the L1 protein of HPV-16.

The polymerase chain reaction (PCR) was used to amplify a region of the gag gene, encompassing the core protein p27, from genomic DNA of cells infected with SIVmac251 (32H isolate). The 767 base pair PCR product was cloned into the bacteriophage M13 and fully sequenced before sub-cloning into the expression vector pUC19. The 30 kilodalton (kDa) fusion protein of lacZ-p27 was expressed as a soluble protein in E. coli JM101 cells and purified to greater than 90% purity by affinity chromatography. The affinity purified product was used in serological and T-cell assays to assess immune function in cynomolgus macaques immunised or challenged with immunodeficiency virus derived material. This reagent and accompanying methods provide valuable assays for monitoring the efficacy of vaccines for SIV as a model for human AIDS.

The human papillomavirus type 16 (HPV-16) E7 gene cooperates with an activated ras oncogene to transform primary rodent cells and is important in the immortalization of cervical keratinocytes. We have generated a series of point mutations within the E7 gene and show that mutation of residues serine 31 and serine 71 affect the phosphorylation of the E7 protein, but do not alter its ability to cooperate with ras. Further mutations which alter cysteine residues in a -Cys-X-X-Cys- motif decrease transformation markedly, although they do not abolish it entirely. All the mutations generated displayed a decreased ability to transactivate the adenovirus E2 promoter. These results show that neither phosphorylation of E7 nor its ability to transactivate are required for transformation by E7.

A rapid, non-radioactive assay for the detection of proviral Simian immunodeficiency virus (SIV) in tissue-culture cells is described. The assay is based on the co-amplification of the SIV env and gag genes by the polymerase chain reaction (PCR). When the gag PCR product is blotted onto a nylon membrane and hybridised to a radioactive oligonucleotide probe, the assay can also be used to detect the SIV gag gene in DNA isolated directly from experimentally infected cynomolgus macaque lymphocytes. This provides a valuable assay for the presence of proviral SIV during animal trials of AIDS vaccines and chemotherapeutics.

A comparison of the humoral response to resistant (NIH) and susceptible (C3H) strains of mice, which reject adult worms at different rates during a primary infection, was made following infection with Trichinella spiralis. The serum concentration of immunoglobulins of the heavy chain classes IgM, IgG1, IgG2, IgG3 and IgA were determined by single radial immunodiffusion. Antibodies of the same immunoglobulin isotypes to biochemically defined, stage specific surface and secreted components of three stages of parasite development were also determined using an isotype specific immuno-coprecipitation assay. Independent variation of the responses of each immunoglobulin isotype was observed. The specific anti-parasite response did not reflect total serum immunoglobulin levels in all immunoglobulin classes, and this is discussed in relation to basic mechanisms of immunoglobulin class switching. Finally a close correlation was observed in resistant (NIH) mice between the production of IgA antibody to surface components of adult worms and accelerated expulsion of this stage of the worm from the gastrointestinal tract. The possible relevance of this IgA response is further indicated by the failure of susceptible mice to synthesise IgA antibodies to the same surface antigens.

The effect of constitutive expression of the c-myc oncogene on the biological properties of cells transformed or immortalized by human papilloma virus type 16 (HPV16) was studied. Whereas transfection of HPV16 alone into primary baby mouse kidney (BMK) cells failed to generate any immortalized cell lines unless the tumor promoter phorbol 12-myristate 13-acetate was present, cotransfection of HPV16 with a plasmid that constitutively expresses murine c-myc (pSVc-myc-1) generated numerous rapidly growing colonies of cells. Cell lines transformed by HPV16 and pSVc-myc-1 did not require phorbol ester or steroid hormones for growth and were tumorigenic in syngeneic immunocompetent mice. Transfection of pSVc-myc-1 into established cell lines transformed by HPV16 and the v-fos oncogene increased the growth rate and saturation density of these lines severalfold, allowed growth in low-serum medium, and abolished the requirement of these cell lines for glucocorticoids or progestogens. Transfection of the EJ-ras oncogene into these lines did not significantly affect any of these properties.

Objective: To compare the long term effects of short term intravenous infusions of iloprost with those of oral nifedipine in patients with Raynaud's phenomenon associated with systemic sclerosis., Design: Double blind, placebo controlled, randomised group comparison., Setting: Dermatology outpatient clinic., Patients: Twenty three patients with Raynaud's phenomenon associated with well documented systemic sclerosis (American Rheumatism Association criteria) and with typical abnormalities in fingernail folds on capillaroscopy., Interventions: Twelve patients were randomised to receive intravenous infusions of iloprost starting at 0.5 ng/kg/min and increased by 0.5 ng/kg/min every 15 minutes to a maximum of 2.0 ng/kg/min for eight hours on three consecutive days with a further single infusion at week 8. Placebo capsules were given concurrently. Eleven patients were randomised to receive nifedipine, starting at 30 mg daily and increased to 60 mg daily after four weeks for another 12 weeks. Infusions of placebo were given in the same manner as the infusions of iloprost. One patient from each group withdrew because of social reasons and three patients receiving nifedipine withdrew because of side effects., End Point: Reduction in number, duration, and severity of attacks of Raynaud's phenomenon, reduction in number of digital lesions, increase in digital blood flow., Measurements and Main Results: Measurements were taken at 0, 4, 8, 12, and 16 weeks. Both regimens produced a reduction in the number, duration, and severity of attacks of Raynaud's phenomenon. The mean (SE) number of digital lesions was reduced with iloprost (from 3.5 (1.6) to 0.6 (0.3] and with nifedipine (from 4.3 (0.8) to 1.4 (0.5] after 16 weeks. Hand temperature and digital and microcirculatory blood flow were increased with iloprost but not with nifedipine., Conclusion: Both iloprost and nifedipine are beneficial in the treatment of Raynaud's phenomenon. With nifedipine, however, side effects are common. Short term infusions of iloprost provide longlasting relief of symptoms, and side effects occur only during the infusions and are dose dependent.

Fifteen patients with primary Raynaud's phenomenon received captopril 25 mg or placebo, three times daily for 6 weeks, in a randomized double-blind cross-over study. Compared with placebo, captopril produced a significant improvement in cutaneous blood flow but did not alter the frequency or severity of attacks of Raynaud's phenomenon.

Biochemically characterised surface and secreted stage specific antigens were used to analyse the response of humans to infection with Trichinella spiralis. Immuno-coprecipitation and SDS-PAGE of labelled proteins were used to dissect the humoral response of Mexican and European infected sera to each component antigen. The results demonstrate that in man, like laboratory rodents, the response to each protein varies independently. In addition, they permit a more rational approach to the development of improved immuno-diagnostic techniques.

Under certain conditions laser Doppler flowmeter (LDF) signals obtained from the finger pulp may appear very similar to those obtained by use of a direct current (dc) photoplethysmograph (PPG). A combined LDF/PPG system was used in conjunction with a circumference strain gauge as an index of volume change to identify the conditions in which the correlation between these signals was good. Simultaneous LDF and dc PPG measurements were made on 10 normal volunteers by using arterial occlusion and on 7 normal subjects by using the Valsalva maneuver at different elevations of the forearm and hand with respect to the midsternum. By altering the elevation of the upper limb the influence of venous filling on each of the signals during these maneuvers was observed. Since the dc PPG signal always appeared similar to the volume change indicated by the circumference strain gauge, it is concluded that the dc PPG signal is related to blood volume change if allowance is made for the effects of blood oxygenation. In circumstances of low venous filling, however, blood volume changes correlate well with blood flow changes, producing the correlation between the dc PPG and LDF traces. The dc PPG signal may be used as a means of monitoring changes in blood flow in the finger only when venous filling is low and the return remains unrestricted. Thus, in investigations using this method, the relative position of the limb with respect to the heart should always be indicated. The LDF method appears to be a reliable indicator of blood flow changes in the microcirculation irrespective of the degree of venous filling.

The Escherichia coli rpsU-dnaG-rpoD operon contains an internal transcription terminator T1 located in the intergenic region between the rpsU and dnaG genes (Smiley et al. 1982). By cloning T1 as a small 127 bp fragment into the terminator probe plasmid pDR720 between the trp operator promoter and the assayable galK gene, it was shown that T1 acts as a strong transcription terminator, comparable in strength to the 3' operon terminator T2. However, an operon sequence that occurs 5' to T1 within the coding region of the rpsU gene and which has homology with the lambda nut site, (Lupski et al. 1983) when placed 5' to T1 in the pDR720 plasmid construct, modifies transcription through T1 allowing expression of the galK gene. This sequence, called the dnaG nut site also modifies the termination activity of the external operon terminator T2. It is proposed that the dnaG nut site is a cis-acting element of an antitermination system in E. coli.

A thorough study of parasite antigens is a prerequisite for control programmes based on protection by vaccination, accurate serodiagnosis and perhaps immune modulation to diminish pathological sequelae. Stage specific surface secreted and somatic antigens may be of particular value in proceeding towards these goals. The design of vaccines is most appropriately focused on surface antigens. With respect to pathology, certain antigens must stimulate humoral and, or cellular immune responses which are responsible for the undesirable immunopathologic consequences of the disease. The ultimate objective, therefore, is identification of those particular antigens followed by appropriate down regulation of the immune system in order to delete such potentially harmful immunological reactions. The relevant illustration presented in this context is an interesting correlation between one particular clinical condition of onchocerciasis ("sowda") and the serological response, defined both in terms of the parasite antigen and an immunoglobulin class restricted antibody response. Current parasitological methods of diagnosis consistently underestimate parasite prevalence. Failure to detect low level patent infections incurs the risk of having a reservoir capable of perpetuating infections. There is, then, an urgent requirement for accurate serodiagnosis, to be used in association with, and for the evaluation of, drug treatment and vector elimination in parasite control programmes. Given the high sensitivity of current immunoassay technology, the only bar to establishing the necessary immunological tests is the choice of suitably specific antibody-antigen systems. Once these are identified, a combination of recombinant nucleic acid biochemistry and hybridoma technology should provide the necessary reagents for inexpensive, robust and specific diagnostic tests. In addition, it may not be many years before the ubiquitous RIA and ELISA technology gives way to the newly developing biosensor systems. Finally, given the sensitivity and specificity of today's nucleic acid hybridization techniques, we may soon expect to see specific identification of infective larvae in their vectors of this, a cloned DNA probe specific for Onchocerca volvulus, and with potential for the detection of infective larvae in blackflies is described.

The effect of captopril on skin response to intradermal injection of bradykinin was investigated by laser Doppler flowmetry (LDF) and weal and flare measurements in this randomised, double-blind, placebo-controlled, cross-over balanced study. Intradermal injections of 1 and 2.5 micrograms of bradykinin and normal saline were made into the forearm skin of six healthy volunteers between 1 and 2 h (t1) and between 3 and 4 h (t2) after either 25 mg captopril or placebo. Skin blood flow outside the induced weal was monitored continuously by LDF for 15 min and the mean LDF values over the last 15 s were used for analysis. Weal and flare sizes were measured at 15 min. On the placebo days, the mean LDF output, weal volume and flare area increased with incremental bradykinin dose. Pre-treatment with captopril significantly increased LDF output following intradermal bradykinin at t1 but not at t2. At both t1 and t2, captopril significantly increased weal volume. There was no significant difference between treatments in flare areas. Skin response following intradermal normal saline, measured by the above parameters, was not affected by captopril. This study showed that captopril potentiated the effects of intradermal bradykinin both with respect to blood flow changes and weal formation. The non-invasive technique of LDF can be used to detect the skin blood flow changes induced by intradermal bradykinin and the potentiation of this effect by captopril. It appears to be a useful and more objective method of quantifying local cutaneous blood flow changes than measurement of flare area.

Intact, viable adults, infective and newborn larvae of Trichinella pseudospiralis were surface labelled with 125I by the chloramine T method and labelled proteins were compared with those obtained from equivalent stages of T. spiralis. Electron-microscope autoradiography determined that labelled proteins were restricted to the cuticle for all stages of both isolates. Comparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE), using thin gradient gel slabs, of proteins obtained from each stage, demonstrated that the profile of surface-labelled proteins of T. pseudospiralis were restricted in number, stage specific, and similar to equivalent proteins of T. spiralis both in size and in their organization into aggregates. The stage-specific profiles of surface-labelled proteins derived from newborn larvae were indistinguishable, but differences were noted between adults and infective larvae of the two isolates. These differences in protein structure were confirmed by two dimensional mapping of tryptic peptides. Stage-specific profiles were also obtained when [35S]methionine biosynthetically labelled secretions of the 3 stages of T. pseudospiralis were compared by SDS-PAGE. Comparison of the profiles obtained with secretions for respective stages of T. spiralis again failed to distinguish newborn larvae, but adults and infective larvae of T. spiralis and T. pseudospiralis displayed a mixture of common and species-specific proteins. These findings are discussed in relation to the different pathology associated with infection with two isolates.

The anti-phosphoryl choline antibody responses of resistant and susceptible strains of mice infected with microfilariae of Dipetalonema viteae or the gastrointestinal nematode parasite Trichinella spiralis were determined. The responses were characterized both in terms of their kinetics and the constituent role played by each immunoglobulin heavy-chain class. Infection with either parasite elicited detectable responses in all strains investigated. However, the kinetics of the response of each heavy-chain class within each strain could vary independently. Furthermore, differences were observed between the responses of inbred mouse strains that differ in their ability to control infection. It was not possible, however, to correlate these differences in the serological response with the ability of these animals to control infection. The results are discussed in relation to attempts to improve serological diagnostic assays.

A temporal relationship has been demonstrated between persisting immune complexes and non-antigen-specific immunodepression. Mice were given intraperitoneal injections of Bordetella pertussis at weekly intervals. After 7 weeks they developed circulating immune complexes, the levels of which increased with continued administration of pertussis. The increase in immune complex levels was accompanied by a diminished primary immune response to intraperitoneally injected sheep erythrocytes (SRBC) as judged by a reduction in their direct and indirect plaque-forming cell response and serum agglutination titres. Spleen cells from immunodepressed pertussis-treated mice were transferred to irradiated normal recipients and displayed a normal response to SRBC. By contrast, spleen cells transferred from normal donors to irradiated pertussis-treated recipients had an impaired response to SRBC. Thus, the immunodepression caused by pertussis treatment is a property of the environment and not the lymphocytes themselves. It is considered that chronic circulating immune complexes induced by pertussis administration may cause non-antigen-specific immunodepression.

The effect of human papillomavirus (HPV) and activated oncogenes on the growth and morphology of primary baby mouse kidney (BMK) cells has been studied. Early region DNA from HPV types 16, 18, 31, and 33, but not type 6, under the transcriptional control of a heterologous, retroviral promoter cooperated with EJ-ras to produce cell lines that gave rise to carcinomas in syngeneic immunocompetent animals. The same HPV constructs, when cotransfected with a plasmid containing sequences from the Finkel-Biskis-Jinkins murine sarcoma virus provirus (v-fos), produced cell lines that were tumorigenic in nude mice. None of the other activated oncogenes tested, including activated c-myc, displayed any activity with HPV in this cotransfection assay. When the heterologous promoter was replaced by the homologous HPV16 promoter, the transforming effect of HPV16 with either EJ-ras or v-fos required the presence of either glucocorticoid or progestogen. Cell lines derived from transfection of HPV16 with either EJ-ras or v-fos required the continued presence of hormones for proliferation.

An improvement in the quality of a photoplethysmography signal derived from a laser Doppler flowmeter probe has been achieved by incorporating an auxiliary fibre in the probe head. This fibre is positioned at an optimum distance from the laser light transmitting fibre and overcomes the problem of high frequency signals which mask the detailed features of the photoplethysmograph pulse when a small fibre separation is used. These two signals may be recorded simultaneously from the same site and a correlation between the Doppler output and the photoplethysmograph amplitude has been demonstrated in the finger. The amplitude is shown to be affected by the relative position of the point of measurement with respect to the heart, a factor which does not appear to influence significantly the Doppler output.

DNA prepared from four isolates of Trichinella was compared by genomic DNA cross-hybridisation, by electrophoresis following restriction endonuclease digestion and by hybridisation studies using a cloned repetitive DNA sequence from T. spiralis. The DNA from T. spiralis, T. nelsoni and T. pseudospiralis isolates was distinct and the interrelationships of these isolates were inferred. In contrast to previous work on T. nativa and T. spiralis, our work suggests that these two isolates are very similar.

The temperature response of the hands to mild cold stress (20 degrees C for one minute) has been measured in 20 normal subjects, 20 patients with reflex sympathetic dystrophy (RSD) and 10 patients with chronic upper limb pain (CULP) of uncertain origin. The results of RSD and CULP groups were significantly (p less than 0.05) different from normal but were indistinguishable. For each patient, 11 variables obtained from the thermal stress test were compared with the normal range. Ten of the RSD group and seven of the CULP group had four or more abnormal variables and were considered to have a thermoregulatory abnormality. The thermal stress test is useful in the objective assessment of RSD. It is non-invasive, patient acceptable and reproducible.

The class-specific antibody responses of 3 strains of mice (C57/Bl10, BALB/C and CBA/N) known to vary in their ability to control the microfilaraemia which follows the subcutaneous transplantation of adult female Dipetalonema viteae has been investigated. The 3 mouse strains showed significant variation (a) in total levels of immunoglobulins and (b) in ability to recognize individual radio-isotope-labelled antigens as measured by coprecipitation. Within each mouse strain it was noted that antigens could vary with respect to the nature of the isotype of the antibody response which they elicited. Furthermore, by comparing results obtained from class-specific coprecipitation with surface ELISA it was found that a similar variation between responses to individual epitopes was also likely. No differences were observed in the humoral response of the 3 mouse strains which could explain the known resistance of the C57/Bl10 strain; reasons for this are discussed.