Detection of active tuberculosis by an MPB-64 transdermal patch: a field study.

The mycobacterial antigen MPB-64 was formulated for delivery in a transdermal patch and used as a diagnostic skin test reagent to detect active tuberculosis (TB) in patients attending a clinic in Manila, The Philippines. The MPB-64 Transdermal Patch was applied to 62 patients, 49 with sputum-positive active disease and 13 who had completed TB chemotherapy, and to 28 non-TB but tuberculin-positive controls. The results were read at 72 h. The sensitivity of the Transdermal Patch was 87.8%, with an efficacy of 92.9% and a specificity of 100%. The 13 TB patients who had completed 6 months of TB chemotherapy showed different reactions to the MPB64 patch test: those who had completed chemotherapy < 4 months before testing were positive; 50% of patients who completed chemotherapy 5 months previously were positive; and those who had completed chemotherapy 7 and 8 months before were negative. All the non-TB controls with positive tuberculin tests were negative to the MPB-64 Transdermal Patch, even at the highest protein dose tested. This test may be a useful method to distinguish active TB patients from TB-infected but asymptomatic individuals. Moreover, the MPB64 Transdermal Patch may be useful to monitor successful chemotherapy.

Liposomal vaccines.

Liposomes have been used therapeutically to deliver drugs to certain anatomical sites. The use of liposomes to deliver antigens, although not a new concept, has received less attention. At least two vaccines of nearly identical liposome base composition to our vaccines have been tested in humans. A malaria vaccine study showed that the liposomal preparation is quite safe: reaction profiles of volunteers receiving the vaccine demonstrated little reactivity and virtually no pyrogenicity (14). The concentration of MPLA in the vaccine was substantially higher (nearly 50,000 times) than the pyrogenic dose of free lipid A. The same vaccine, but different antigen (gp120, an HIV protein), was tested in volunteers and had the same lack of toxicity (27). In both studies, antibodies and cytotoxic cells specific for the respective antigens were produced. We have several subunit vaccines under development for infectious diseases (gram negative sepsis, fungal infections, protozoan infections), metabolic disorders (hypercholesterolemia, diabetic retinopathy, macular degeneration), and neoplastic diseases (multi-drug resistant cancer, primary and metastatic tumors, and angiogenic hyperproliferative disorders). In each case, one or more antigens were identified that might be useful in immunologic control of biologic proliferation (i.e., pathogen or tumor growth, rise in serum cholesterol, growth of blood vessels). We anticipate that at least one of these vaccines will be ready for testing in humans in the next calendar year.

The use of ampligen alone and in combination with ganciclovir and coumermycin A1 for the treatment of ducks congenitally-infected with duck hepatitis B virus.

Ampligen, a known immunomodulator and interferon inducer, was used alone and in combination with other antiviral agents to treat ducks congenitally-infected with duck hepatitis B virus. These antiviral agents included the conventional nucleoside analogue ganciclovir and the prokaryotic DNA gyrase B inhibitor coumermycin A1. When used alone, ampligen decreased the amount of serum and liver viral DNA, but had no effect on circulating duck hepatitis B surface antigen (DHBsAg). In combination with ganciclovir, the antiviral effect appeared at least additive with a greater inhibition of viral DNA replication within the liver. The combination of ampligen with coumermycin A1 also resulted in inhibition of viral replication but to a lesser extent than ampligen alone. When all three agents were used together, viral DNA replication was again inhibited, but as with previous treatment regimes, serum DHBsAg levels remained unchanged. At the end of the treatment period for all regimes, analysis of viral DNA forms in the liver showed that the viral relaxed circular and supercoiled DNA forms had persisted. Within 1 week of cessation of therapy, viral replication had often returned to pre-treatment levels. Interferon-like activity was detected in the sera of the majority of the treated ducks during the ampligen therapy, but no clear relationship between the presence of interferon and antiviral effect could be established. These observations in the duck hepatitis B model may provide a rational basis for the use of combinations of antiviral and immunomodulatory regimes for the management of chronic hepatitis B infection in man.

Clinical, immunological, and virological effects of ampligen, a mismatched double-stranded RNA, in patients with AIDS or AIDS-related complex.

10 patients with the acquired immunodeficiency syndrome (AIDS), AIDS-related complex (ARC), or lymphadenopathy syndrome (LAS) were given 200-250 mg ampligen, a mismatched double-stranded (ds) RNA with in-vitro antiviral activity against human immunodeficiency virus (HIV), twice a week for up to 18 weeks, without side-effects or toxicity. In all 9 patients who were positive for HIV RNA in peripheral blood mononuclear cells before therapy, levels became undetectable between days 10 and 40 of the start of therapy. 6 of the 7 patients with ARC or LAS also showed a progressive reduction in HIV load as measured by co-culture assays. All 10 patients had augmentation of delayed-type hypersensitivity skin reactions. Other changes noted during ampligen therapy included an increase in or maintenance of numbers of helper-inducer T lymphocytes, improvements in HIV-related symptoms, rises in titre of neutralising antibodies against HIV, and restoration of proper functioning of the natural lymphocyte antiviral dsRNA-dependent (2'-5'-oligoadenylate/RNA-ase L) pathway. Thus, in the short term, ampligen seems to have the dual ability to restore immunological function and to control HIV replication.

Chromatin structure of the cytochrome P-450c gene changes following induction.

The chromatin structure of cytochrome P-450c and P-450d genes, which in the liver are highly inducible by 3-methylcholanthrene, was studied in normal and carcinogen-treated rats by using a cDNA probe specific for P-450c and a genomic probe that recognizes both genes. Digestion with micrococcal nuclease revealed that the active genes are not present in the typical 200 base pair nucleosomal structure. Gene induction is associated with a rearrangement of the nuclear organization of the genes. By use of indirect end-label hybridization, three DNase I hypersensitive sites were mapped, one in the 5'-terminal region and two in the 3' region of the P-450c gene. Gene induction, by treatment with 3-methylcholanthrene, changes the location of the DNase I site present in the 5' region without affecting the sites present in the 3' region. Rat thymus chromatin does not contain these DNase I hypersensitive sites, suggesting that, in the liver, the chromatin structure is altered so as to allow tissue-specific expression of the P-450c gene. The chromatin structure of the highly inducible P-450c gene is compared to that of the P-450m gene, which is induced to a significantly smaller extent and is constitutively expressed.

Chromatin structure of a 3-methylcholanthrene-induced cytochrome P-450 gene.

Plasmids carrying fragments of a cytochrome P-450 gene, inducible by 3-methylcholanthrene, were used to study the chromatin structure of this gene in the liver of normal and carcinogen-treated rats. Digestion with micrococcal nuclease revealed that the gene is not present in the typical 200 base pair nucleosomal structure. By use of indirect end-label hybridization, four DNase I hypersensitive sites were mapped in the 5'-terminal region of the gene. An S1 nuclease sensitive site is located close to a DNase I site. Gene induction by treatment with 3-methylcholanthrene does not result in detectable changes in the DNase I hypersensitive sites. Rat thymus chromatin does not contain DNase I hypersensitive sites in the P-450 gene, suggesting that in the liver the chromatin structure is altered so as to allow tissue-specific expression of the gene. This paper is the first study on the chromatin structure of a gene coding for a member of the cytochrome P-450 family of enzymes. The implications of our results to the understanding of gene regulation of the P-450 genes are discussed.

The intracellular distribution and function of the high mobility group chromosomal proteins.

This brief review provides a framework for discussing current approaches being used to determine the cellular localization and function of the high mobility group chromosomal (HMG) proteins. The four main constituents of this group (HMG 1, 2, 14, 17) are present in all four eukaryotic kingdoms, have a relatively well conserved primary sequence and contain several functional domains which enable them to interact with DNA, histones and other components of the genome. The evolutionary conservation in the primary and tertiary structure as well as the observed correlations between cell phenotype and quantitative changes in protein levels and in post-synthesis modifications suggests that these proteins are components obligatory for proper cellular function. Proteins HMG 1, 2 are DNA-binding proteins which can distinguish between various types of single-stranded regions of the genome. Proteins HMG 14, 17 may be involved in maintaining specific chromatin regions in particular conformations. The data available presently suggests that these proteins are important structural elements of chromatin and chromosomes.

Localization of HMG chromosomal proteins in the nucleus and cytoplasm by microinjection of functional antibody fragments into living fibroblasts.

We have used microinjection and cell fractionation to localize the chromosomal high mobility group proteins (HMG) in human fibroblasts. Electrophoretic analysis of nuclear and cytoplasmic fractions from the fibroblasts indicates that the concentration of HMG-1,2 in the cytoplasm is 2.9 times larger than in the nucleus indicating that the majority of the cellular HMG-1,2 is present in the cytoplasm. In contrast, HMG-17 remains predominant in the nuclear fraction. We conclude that the cellular distribution of HMG-1,2 is significantly different from that of HMG-17. To avoid possible artifacts due to cell fractionation, fluoresceinated HMG-1 and HMG antibodies were microinjected into living fibroblasts. The cellular distribution of the injected proteins was monitored using fluorescent microscopy. Fluoresceinated HMG-1 microinjected into the cytoplasm moves very rapidly into the nucleus and concentrates in the nucleolus of living human fibroblasts. However, some control non-nuclear proteins also migrated into the nucleus raising the possibility that exogenous injected proteins do not always distribute in the same pattern as the endogenous proteins. The localization of microinjected F(ab)2 fragments derived from anti-HMG-1 was compared to that of microinjected F(ab)2 derived from anti-histones. Whereas the anti-histone F(ab)2 when injected into the cytoplasm migrated into the nucleus, the anti-HMG-1 F(ab)2 remained in the cytoplasm. Microinjection of anti-HMG-17 and anti-histone inhibited transcription in living cells, anti-HMG-1,2 did not. We conclude that HMG-1,2 proteins are present in both the nucleus and cytoplasm of living fibroblasts.

Functional histone antibody fragments traverse the nuclear envelope.

Factors important in the translocation process of proteins across the nuclear membrane were studied by microinjecting either fluoresceinated nonimmune IgG and F(ab)2 or the corresponding molecules, prepared from antisera to histones, into the nucleus and cytoplasm of human fibroblasts. Intact IgG from both preparations remained at the site of injection regardless of whether it was injected into the nucleus or the cytoplasm. In contrast, nonimmune F(ab)2 distributed uniformly throughout the cell. The F(ab)2 derived from affinity-pure antihistone moves into the nucleus after cytoplasmic injection and remains in the nucleus after nuclear microinjection. The migration of the antihistone F(ab)2 into the nucleus results in inhibition of uridine incorporation in the nuclei of the microinjected cells. We conclude that non-nuclear proteins, devoid of specific signal sequences, traverse the nuclear membrane and accumulate in the nucleus provided their radius of gyration is less than 55A and the nucleus contains binding sites for these molecules. These findings support the model of "quasibifunctional binding sites" as a driving force for nuclear accumulation of proteins. The results also indicate that active F(ab)2 fragments, microinjected into somatic cells, can bind to their antigenic sites suggesting that microinjection of active antibody fragments can be used to study the location and function of nuclear components in living cells.

Inhibition of transcription in somatic cells by microinjection of antibodies to chromosomal proteins.

The in vivo function of defined chromosomal proteins was examined by microinjecting purified antibody and antibody fragments into living fibroblasts. The involvement of histones and chromosomal high mobility group proteins HMG-1, 2, and 17 in transcription was visualized by studying the [3H]uridine incorporation in KD human fibroblasts after microinjection of fluoresceinated antibodies to these proteins. Nuclear uridine incorporation was not affected by microinjection of control antibodies or by the presence of immune complexes formed after microinjection of antibodies to chromosomal proteins that are not involved in transcription. In contrast, injection of anti-histone IgG, F(ab')2, or Fab and anti-HMG-17 IgG causes a significant reduction in transcription. The reduction is proportional to the amount of antibody introduced into the cell. We conclude that histones and protein HMG-17 are present on transcribed regions of the genome and that passage of RNA polymerase along the chromatin fiber is prevented by antibody binding to these proteins.

Autoantibodies to nucleosomal proteins: antibodies to HMG-17 in autoimmune diseases.

The relative amounts of autoantibodies against defined nucleosomal proteins present in serums from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and mixed connective tissue disease (MCTD) have been examined by an enzyme-linked immunoassay. Autoantibodies to nucleosomal proteins were detected in 45 percent of the patients with SLE, 18 percent of the MCTD patients, and none of the RA patients. The results suggest that, in SLE, antibodies are formed against a subset of nucleosomes which contain protein HMG-17.

Micrococcal nuclease cleavage of chromatin displays nonrandom properties.

A statistical analysis of the products of digestion of chicken erythrocyte chromatin by micrococcal nuclease was used to test for randomness of the cutting process. DNA fragment size classes corresponding to mononucleosome, dinucleosome, trinucleosome, tetranucleosome, and all fragments larger than tetranucleosome were evaluated. In every case, fragments in the mononucleosome and greater-than-tetranucleosome classes were produced in excess of the level expected on the basis of random cleavage while those in the dinucleosome-tetranucleosome classes exhibited a shortage. The pattern of nonrandomness appears to depend on substrate size: the magnitude of deviations from randomness was large when substrates of genomic size are compared with polynucleosomal segments whereas the direction of deviation is identical. Nonrandomness was independent of ionic conditions known to affect the state of chromatin condensation and also appeared to be unaffected by depletion of histones H1 and H5. The possible universality of nonrandom cleavage was suggested when other data from the literature was analyzed. Some possible mechanisms to account for this property are discussed.

Adhesion of particulate specimens to support films for electron microscopy: a model system for assessing the surface properties of support films, and its application to chromatin particles.

Silica microspheres bearing a known surface charge were used to test the adhesive properties of support films and support film treatments commonly used in the electron microscopy of particulate specimens. Adhesion was strongly correlated with surface charge, negatively charged microspheres binding well only to positively charged support films and vice versa in solutions of low ionic strength. This charge dependency could be overcome by increasing the ionic strength to about 100 mmol with monovalent cations; under these conditions, it was not necessary to provide an oppositely charged film surface to obtain adhesion. Chromatin particles (nucleosomes) which have a net negative charge, behaved very much like the negatively charged silica with respect to adhesion, confirming that the microspheres provided an accurate indication of support film surface properties. The chromatin particles showed dramatic structural changes under conditions when adhesion was either poor, or very strong, indicating the need for careful selection of binding conditions for delicate biological specimens. A new and simple method for pretreating carbon films to improve adhesion was developed, and a preliminary account of this technique is presented.