Broadband up-conversion at subsolar irradiance: triplet-triplet annihilation boosted by fluorescent semiconductor nanocrystals.

Conventional solar cells exhibit limited efficiencies in part due to their inability to absorb the entire solar spectrum. Sub-band-gap photons are typically lost but could be captured if a material that performs up-conversion, which shifts photon energies higher, is coupled to the device. Recently, molecular chromophores that undergo triplet-triplet annihilation (TTA) have shown promise for efficient up-conversion at low irradiance, suitable for some types of solar cells. However, the molecular systems that have shown the highest up-conversion efficiency to date are ill suited to broadband light harvesting, reducing their applicability. Here we overcome this limitation by combining an organic TTA system with highly fluorescent CdSe semiconductor nanocrystals. Because of their broadband absorption and spectrally narrow, size-tunable fluorescence, the nanocrystals absorb the radiation lost by the TTA chromophores, returning this energy to the up-converter. The resulting nanocrystal-boosted system shows a doubled light-harvesting ability, which allows a green-to-blue conversion efficiency of ∼12.5% under 0.5 suns of incoherent excitation. This record efficiency at subsolar irradiance demonstrates that boosting the TTA by light-emitting nanocrystals can potentially provide a general route for up-conversion for different photovoltaic and photocatalytic applications.

New mutations in the neuronal ceroid lipofuscinosis genes.

Thirty-eight mutations and seven polymorphisms have recently been reported in the genes underlying the neuronal ceroid lipofuscinoses (NCLs) including 11 new mutations described here. A total of 114 mutations and 28 polymorphisms have now been described in the five human genes identified which cause NCL. Thirty-eight mutations are recorded for CLN1/PPT; 40 for CLN2/TTP-1, 31 for CLN3, four for CLN5, one for CLN8. Two mutations have been described in animal genes (cln8/mnd, CTSD). All mutations in NCL genes are contained in the NCL Mutation Database (http://www.ucl.ac.uk/NCL).

Phenotype-genotype correlation in eight patients with Finnish variant late infantile NCL (CLN5).

The authors analyzed the clinical phenotype, including MRI, of eight patients with Finnish variant late infantile neuronal ceroid lipofuscinosis (vLINCLFin; CLN5; MIM256731). Although the four known mutations, including one novel mutation identified in this study, have very different consequences for the predicted polypeptide, none of them results in an atypical phenotype, as has been reported in other forms of NCL. Thus, it seems likely that each mutation severely disturbs the normal function of the CLN5 protein.

Transcript identification on the CLN5 region on chromosome 13q22.

Our efforts to clone the CLN5 gene, mutated in a severe children's brain disease, variant late infantile neuronal ceroid lipofuscinosis (vLINCL, MIM256731), resulted in large-scale sequencing of genomic clones flanking the critical chromosomal region on 13q22. Computational and traditional transcript identification analyses of the resulting sequence were used to identify the disease gene. In addition to the identification of the CLN5 gene, this effort produced a large amount of genomic sequence data. Here, we report a transcription map of the 107 kb sequence in the CLN5 region, based on traditional and computational gene identification strategies. Several transcripts were identified in this sequence. Queries against the database of expressed sequence tags proved to be the most powerful tool for gene identification from large-scale sequence.

Coreceptor usage and RANTES sensitivity of non-syncytium-inducing HIV-1 isolates obtained from patients with AIDS.

OBJECTIVES: The biologic phenotype of HIV-1 primary isolates obtained from approximately 50% of patients who progress to AIDS switches from non-syncytium-inducing (NSI) to syncytium-inducing (SI). We evaluated possible associations between virus coreceptor usage, sensitivity to inhibition by beta-chemokines, and disease progression of patients who continue to yield NSI isolates after developing AIDS. STUDY DESIGN/METHODS: Sequential virus isolates were analyzed for biologic phenotype using the MT-2 cell assay, for sensitivity to beta-chemokines using RANTES inhibition, and for coreceptor usage using U87.CD4 and GHOST.CD4 cells expressing different chemokine/orphan receptors or donor peripheral blood mononuclear cells (PBMC) defective in CCR5 expression. In addition, the env V3 region was sequenced and the length of the V2 region determined. RESULTS: All NSI isolates, regardless of patient status at time of isolation, were dependent on CCR5 expression for cell entry. Furthermore, there was no indication of broadened coreceptor usage of NSI isolates obtained from persons with late-stage AIDS. A majority of NSI isolates remained RANTES sensitive; however, virus variants with reduced sensitivity were observed. The V2 lengths and the V3 sequences exhibited no or minor changes at analysis of sequential NSI isolates. CONCLUSIONS: Our data suggest that NSI isolates obtained from AIDS patients remain CCR5 dependent (ie, R5) and, in many cases, also remain sensitive to RANTES inhibition. However, virus variants with decreased sensitivity to RANTES inhibition may evolve during disease progression, not only as a result of a switch from NSI to SI but also in patients who develop AIDS while continuing to maintain R5 isolates.

Indicators of T-cell activation: correlation between quantitative CD38 expression and soluble CD8 levels in asymptomatic HIV+ individuals and healthy controls.

Increased activation of CD8+ T cells, particularly increased expression of CD38 antigen, has been shown to strongly correlate with progression of human immunodeficiency virus-positive (HIV+) individuals to acquired immunodeficiency syndrome (AIDS) and death. As part of a study evaluating responses to a recombinant gp160 vaccine, we have used quantitative three-color flow cytometry (QFCM) to further investigate the relationships among several measures of lymphocyte activation/immunological status. Parameters evaluated included 1) absolute circulating counts for the major lymphocyte phenotypes (T, B, NK) and selected activated/regulatory subsets believed to have clinical value in the monitoring of patients with HIV infection; 2) level of CD38 expression (antibody-binding capacity [ABC]) on the lymphocyte subsets defined by CD8, CD38, and HLA-DR; and 3) serum levels of soluble CD8. CD8+DR+CD38+ counts were found to be markedly increased (approximately 10-fold) in HIV+ individuals, whereas CD4+CD45RA+ counts were markedly decreased (approximately 5-fold). We confirmed previous reports that CD38 expression on CD8 T cells (here reported as CD38 ABC) are increased in asymptomatic HIV+ individuals as compared with healthy controls, and further found that CD38 ABC was elevated approximately 2-fold on CD8+DR+ cells as compared with CD8+DR- cells in healthy controls, and almost 2-fold further elevated on CD8+DR+ cells in HIV+ individuals compared with CD8+DR+ cells in healthy controls. In agreement with previous studies, we found increased serum CD8 levels (sCD8) and increased CD8+DR+ counts in asymptomatic HIV+ individuals. However, when sCD8 was expressed relative to CD8+DR+ cell counts (RsCD8), this index was found to be significantly decreased in HIV+ individuals. Although CD38 ABC on CD8+DR+ cells showed no correlation with sCD8, it was significantly correlated with RsCD8 in both HIV+ and HIV- individuals. Absolute lymphocyte counts were strongly correlated with both CD38 ABC and RsCD8 in HIV+ individuals. However, CD4 counts were correlated with CD38 ABC (but not RsCD8) in HIV+ patients and with RsCD8 (but not CD38 ABC) in HIV-controls. Our results suggest that QFCM is significant in understanding the role of CD8+DR+CD38+ cells in processes such as lymphocyte homeostasis and HIV-induced CD4-cell depletion.

CLN5, a novel gene encoding a putative transmembrane protein mutated in Finnish variant late infantile neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses (NCLs) represent a group of common recessive inherited neurodegenerative disorders of childhood, with an incidence of 1:12,500 live births. They are characterized by accumulation of autofluorescent lipopigments in various tissues. Several forms of NCLs have been identified, based on age at onset, progression of disease, neurophysiological and histopathological findings and separate genetic loci. All types of NCL cause progressive visual and mental decline, motor disturbance, epilepsy and behavioral changes, and lead to premature death. One of the subtypes, Finnish variant late infantile neuronal ceroid lipofuscinosis (vLINCL; MIM256731) affects children at 4-7 years of age. The first symptom is motor clumsiness, followed by progressive visual failure, mental and motor deterioration and later by myoclonia and seizures. We have previously reported linkage for vLINCL on chromosome 13 (ref. 5) and constructed a long-range physical map over the region. Here, we report the positional cloning of a novel gene, CLN5, underlying this severe neurological disorder. The gene encodes a putative transmembrane protein which shows no homology to previously reported proteins. Sequence analysis of DNA samples from patients with three different haplotypes revealed three mutations; one deletion, one nonsense and one missense mutation, suggesting that mutations in this gene are responsible for vLINCL.

Blood cortisol and dehydroepiandrosterone sulphate (DHEAS) levels and CD4 T cell counts in HIV infection.

OBJECTIVE: Blood cortisol and dehydroepiandrosterone sulphate (DHEAS) levels, and CD4 T cell counts were assessed in a group of 44 patients with HIV infection (17 asymptomatic and 27 symptomatic). The steroid levels were compared to those in 80 healthy subjects. RESULTS: The mean cortisol level did not differ between the HIV patients and controls. However, a broad variability existed among the patients; thus, asymptomatic HIV patients revealed a significantly higher mean level than the controls (348 nmol/l vs. 280 nmol/l; p < 0.01). Furthermore, 20 patients had levels above, 16 within, and 8 below the confidence limits of the cortisol levels in controls. This variability might reflect differences in adrenocortical responses to psychological stress and adaptive reactions among patients with HIV infection. The mean DHEAS was markedly lowered in the patient group (1450 nmol/l in patients vs. 3300 nmol/l in controls; p < 0.001). A decrease below the confidence limits of the controls existed in 41 (93%) of the 44 patients. A significant correlation was also found between the low DHEAS levels and low CD4 T cell counts in the patients (p < 0.01), while no such correlation existed for cortisol. CONCLUSION: Whether low DHEAS levels might contribute to some of the pathophysiologic features and/or symptoms seen in HIV infection needs to be investigated.

Immunological and virological interactions in patients receiving passive immunotherapy with HIV-1 neutralizing monoclonal antibodies.

Mouse monoclonal antibodies with high human immunodeficiency virus type 1 (HIV-1) neutralizing titers were used for passive immunotherapy of eleven late-state HIV-infected patients. In five patients the serum level of the core protein p24 decreased, while in five cases it remained unchanged. The level of viral RNA in plasma as measured by quantitative polymerase chain reaction (PCR) decreased in four cases, was stable in another four, and increased in three cases. An anti-mouse (HAMA) response developed in eight patients and anti-idiotypic antibodies appeared in six. Immune complexes that formed in patient sera during the treatment were shown to contain mostly envelope glycoprotein gp120 which decreased in nine of the eleven treated patients toward the end of treatment. Antibodies inhibiting gp120 binding to CD4 became detectable or increased in six patients during immunotherapy. Serology of the HIV-1 V3 region was studied for both the HIV-1 IIIB and MN strains with no or very small changes in titer or avidity after treatment. No change in neutralizing titers to strain HTLVIIIB was observed in serum samples collected before and after treatment was terminated. In nine of the eleven patients stimulation of the T lymphocytes to proliferate in vitro when activated by phytohemagglutinin (PHA) was shown to be increased compared to before treatment. Increased T-cell proliferation was also noted with several antigens such as HIV-1 recombinant antigens, cytomegalovirus (CMV), tetanus toxoid (TT), and purified protein derivate of mycobacterium tuberculosis (PPD). These findings indicate a decreased total gp120 content in serum, permitting better T-cell activation.

HIV antigen-reactive T cells detected by antigen-induced interferon gamma secretion.

The T cell repertoire to human immunodeficiency virus (HIV) was studied in HIV-infected patients of different clinical stages by the detection and enumeration of cells that secreted interferon gamma (IFN-gamma) in short-term cultures of blood mononuclear cells after stimulation in vitro with the HIV recombinant antigens pB1, p121, p24-15, gp160bac, and the HIV V3 loop peptide. T cell reactivities to cytomegalovirus (CMV) and Mycobacterium tuberculosis-purified protein derivative (PPD) were examined in parallel. Among 29 patients with HIV infection, 48% had blood cells recognizing one or more of the five HIV antigens. The mean numbers of HIV antigen-reactive T cells varied between 1/approximately 6000 blood cells for pB1 and 1/approximately 20,000 cells for p24-15. None of the five HIV antigens studied was identified as an immunodominant T cell epitope in HIV infection. T cells from 20% of the patients responded to all five HIV antigens in parallel, but the antigen preferentially recognized varied from patient to patient. Those with more advanced disease had a tendency to lower numbers of HIV antigen-reactive T cells. Most HIV-infected patients had both CMV- and PPD-reactive T cells, but numbers were significantly lower in more advanced disease. It should be possible to adopt the present method to evaluate fine specificities of the T cell repertoire to other antigens and to study the involvement of other cytokines besides IFN-gamma, for example, the Th2 cell-related cytokine interleukin 4.

HIV infection leads to differential expression of T-cell receptor V beta genes in CD4+ and CD8+ T cells.

OBJECTIVE: To analyse variation in T-cell receptor (TCR) V beta gene expression in T cells in HIV-infected individuals. DESIGN: Because there are very few monoclonal antibodies available for studying TCR V beta gene expression, we used polymerase chain reaction (PCR) to analyse the TCR V beta repertoire in HIV-infected individuals using specific primers for 20 distinct families of TCR V beta. METHODS: Evaluation of TCR V beta gene expression in peripheral blood from HIV-1-infected individuals [two in Centers for Disease Control (CDC) stage II, five in CDC stage III and four in CDC stage IV]. Complementary DNA was produced from CD4+ and CD8+ T cells, amplified by PCR and analysed after Southern blotting and hybridization with a C beta-specific oligonucleotide probe. RESULTS: V beta gene expression was dramatically modified, especially in AIDS patients. The CD4+ T-cell subset showed both overexpression (V beta 2) and deletions or underexpression (V beta 9-V beta 20), whereas these gene segments were expressed normally in the CD8+ subset. Only V beta 3 was deleted or underexpression in both CD4+ and CD8+ populations in AIDS patients. CONCLUSIONS: HIV-1 infection induces CD4+ T-cell deficiency, both in total numbers and by causing a paucity of select V beta gene expression in this subset. In addition, the V beta 3 gene family was deleted or underexpressed was observed in both CD4+ and CD8+ T-cell subsets from patients in CDC stage IV. These results are compatible with changes in V beta gene expression known to occur under the action of endogenous or exogenous superantigens.

HIV-1 reverse transcriptase inhibiting antibody titer in serum: relation to disease progression and to core-antibody levels.

A new assay for detecting inhibition of reverse transcriptase activity (the RT-i REA) was developed. This assay was standardized for screening serum samples for reverse transcriptase inhibiting antibodies (RT-iAb). High specificity (100%) and sensitivity (greater than 98%) were achieved with samples from HIV-negative individuals and HIV-infected individuals. The RT-i REA was also used in a study of the titers of RT-iAb in serum samples obtained from 33 HIV-infected homosexual men. The results confirmed the relation between decreasing RT-iAb levels and progression to late stages of the disease. Furthermore, a falling RT-iAb titer was observed in 14 of 15 individuals experiencing periods of severe clinical symptoms attributed to HIV-activity. In 7 of the patients the decline in RT-iAb titer began prior to severe clinical symptoms. The fall in RT-iAb titer also correlated with a reduction in core Ab level. The core Ab level has previously been reported to be a disease progression marker with considerable prognostic value. However, whereas all patients were positive for RT-iAb, 8 of the 33 patients did not have detectable core Ab. The use of RT-iAb titer as a marker of disease progression is discussed.

CD4-mimicking antibodies in HIV-positive and normal human sera.

An earlier study showed that approximately 5% of HIV-seropositive human sera contain gp120 antibodies that mimic CD4, seen as anti-idiotypic to the CD4 monoclonal T4.2. The present study shows the existence of a second type of CD4-mimicking gp120 antibody, which specifically binds to the CD4 monoclonal, OKT4A. This anti-idiotypic antibody to OKT4A is less frequent than the antibody reacting with T4.2. In two patients studied, this OKT4A binding antibody seems to appear as early during infection as the anti-idiotypic antibody to T4.2; however, the concentration varies more with time. In order to evaluate the possible clinical relevance of these CD4-mimicking antibodies, sera from 208 HIV-seropositive and 204 healthy seronegative individuals were screened. There was a significantly higher frequency of anti-idiotypic antibodies to T4.2 among HIV-positive individuals compared to healthy controls (p = 0.05). However, no correlation was found between the presence of CD4-mimicking antibodies and clinical classification of HIV-infected individuals.

Absence of a clinical correlation for complement-mediated, infection-enhancing antibodies in plasma or sera from HIV-1-infected individuals. Multicenter AIDS Cohort Study Group.

Neutralizing and complement-mediated infection-enhancing antibodies to HIV-1 were measured in sera or plasma from 54 HIV-1-positive individuals at various stages of disease, and from an additional 36 HIV-1-positive individuals for whom no clinical data were available. Antibodies were measured in microtiter infection assays utilizing MT-2 cells and the IIIB strain of HIV-1. The frequency of detection of both types of antibodies was identical, being 77 out of 90 cases (86%). Neutralizing and infection-enhancing antibodies were not always found together, and in four cases both were undetectable. No correlation was found between titers of either type of antibody and stage of disease. Furthermore, titers of infection-enhancing antibodies at early stages of disease did not predict rate of disease progression.

HIV reverse transcriptase inhibiting antibodies detected by a new technique: relation to p24 and gp41 antibodies, HIV antigenemia and clinical variables.

A new assay for HIV reverse transcriptase activity inhibiting antibodies (RTI-ab) was used for the analysis of a large collection of sera sampled before and after confirmation of HIV infection. In this assay HIV-RT was preincubated with diluted serum, after which residual RT activity was determined by a technique using a template coupled to macrobeads and 125I-lodo-deoxyuridine-triphosphate as the tracer-substrate. Of the 936 sera analysed, 818 were found positive for RTI-ab, and 824 were positive in Western blot (Wb). The prevalence of RTI-ab compared to Wb was therefore 99.3%. The corresponding figure for 930 sera analysed for envelope-ab, i.e., gp41-ab, was 823 positive, and of these 930 sera 815 were Wb positive, giving a comparative prevalence of 101%. In contrast, only 678 samples of 993 analyzed for core ab, i.e., p24, were positive, giving a prevalence of 77.0% as 880 of these samples were Wb positive. Thus, RTI-ab was as prevalent as gp41-ab, and although the analyses of RTI-ab amounts in different stages showed decreasing levels in stage IV compared to stages II or III, all of the sera except 1 were found positive in stages III and IV. Further, it was found that both the few RTI-ab negative samples in stage II and the few RTI-ab positive samples among Wb negative sera were sampled in connection with seroconversion. The specificity of the RTI-ab assay was 100% in a test of 200 serum samples from HIV negative blood donors. It was concluded that RTI-ab analyses can be made highly sensitive and specific and useful for studies of HIV infection.

Serum levels and catabolism of 3'-azido-3'-deoxythymidine in vivo measured using a specific radioimmunoassay.

The thymidine analogue 3'-azido-3'-deoxythymidine is an effective inhibitor of HIV replication in vitro and is used in the treatment of acquired immunodeficiency syndrome. We report here upon a rapid sensitive radioimmunoassay for the detection of azidothymidine in serum or plasma. The assay is simple to perform and levels as low as 1 ng azidothymidine/ml can be detected. The assay is specific for azidothymidine and shows almost no cross-reaction with closely related nucleoside analogues or with naturally occurring nucleosides. Using this radioimmunoassay we were able to measure the azidothymidine levels in the serum of monkeys and acquired immunodeficiency syndrome patients treated with AZT. Individual variation in the peak serum level and clearance rate of azidothymidine were seen, which emphasizes the need to tailor the dose to the individual.

Carcinoembryonic antigen in cytological specimens of urothelial carcinoma.

A peroxidase-antiperoxidase (PAP) technique was developed for the detection of carcinoembryonic antigen (CEA) in urothelial transitional cells of 52 bladder cancer patients. The percentage of CEA-containing malignant cells varied from 10% to 100%. As a mean, 65% of the malignant cells stained for CEA, while the corresponding figure for benign-looking cells was 24%. The results were compared with cytological evaluations, flow cytophotometric results, and immunofluorescent (IF) staining for CEA. With increasing malignancy, more CEA was detected with the PAP technique, whereas the IF technique failed to show this trend. 18 of 20 malignant-tumors had an aneuploid DNA pattern. The two diploid cases were moderately well differentiated. Samples from bladders with heavy inflammation should be avoided in the PAP technique, since the unspecific staining of granulocytes disturbed a correct evaluation of the transitional cells. The PAP technique used on cytological material is recommended for antigen determinations, since good morphology is obtained.

Short-term prognosis of bladder cancer. Evaluation with urinary CEA.

The parameters U-CEA and stage (T) appear to be good predictors of survival in bladder carcinoma during the first 12 months after diagnosis; calculated with the Cox regression model in a multivariate way. Calculated separately in a bivariate manner 0-12 months after diagnosis, U-CEA and T were highly significant predictors for survival. G did not add to the predictors for survival. Grade (G) did not add to the predictive power during the first 12 months but became more informative a year and later after diagnosis.

Possibilities for an extended classification of bladder cancer.

Patients with bladder cancer were evaluated for T-class, histo-pathological grade and U-CEA (urinary carcinoembryonic antigen) before treatment and the cytological picture 4 months after treatment. Previous work has shown that these variables are not significantly intercorrelated. Scores were computed, consisting of the sums of these (dichotomized) variables. In a statistical analysis with the life-table technique, the scored variables have been log-rank tested for a prognostic trend. In 155 patients, p for symptom-free survival between subgroups with low and high scores was 0.0019 and for relative survival 0.0005. This implies that a combination of variables may have predictive value in bladder cancer.