Immunization with a soluble recombinant HIV protein entrapped in biodegradable microparticles induces HIV-specific CD8+ cytotoxic T lymphocytes and CD4+ Th1 cells.

One of the major obstacles to the development of successful recombinant vaccines against human immunodeficiency virus (HIV) and other intracellular pathogens is the identification of a safe and effective vaccine delivery system for the induction of cell mediated immunity with soluble protein antigens. In this study it was demonstrated that immunization with a recombinant HIV envelop (env) protein entrapped in biodegradable poly(lactide-co-glycolide) (PLG) microparticles induced consistent HIV-specific CD4+ and CD8+ T-cell responses in mice. Major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL) responses were detected following a single systemic immunization with gp120 entrapped microparticles and when given by the intranasal (i.n.) route induced HIV-specific CD8+ CTL and secretory IgA. Furthermore immunization with gp120 entrapped in microparticles generated CD4+ T cells that secreted moderate to high levels of IFN-gamma. Therefore, PLG microparticles are a safe and effective means of delivering antigen to the appropriate processing site for the generation of class I-restricted CTL, and are also capable of inducing Th1 cells.

Preparative separation of foreign antigens for highly efficient presentation to T cells in vitro.

A method is described for the separation and purification of proteins from complex mixtures of foreign antigens in a form suitable for stimulating T cells in vitro. The technique involves electrophoretic separation of proteins followed by elution, concentration and adsorption of the polypeptide subunits to latex microspheres. Alternatively, where a specific antibody is available, proteins may be affinity-purified from a heterogeneous mixture of antigens, using antibody-coated latex microspheres. Nanogram quantities of protein coupled to latex were shown to be highly efficient stimulators of antigen-specific T cells as tested by in vitro proliferation and cytokine release assays. The utility of this technique was demonstrated using poliovirus capsid proteins separated by SDS-polyacrylamide gel electrophoresis (PAGE) and coupled to latex microspheres for specificity analysis of T cell clones. Antigen reactivity of the T cell clones was confirmed using recombinant baculoviruses expressing individual poliovirus proteins. Furthermore, recombinant proteins coupled to latex microspheres were used for efficient stimulation and in vitro propagation of T cell clones specific for the simian immunodeficiency virus (SIV) envelope (env) protein. Although the technique is illustrated in this report using viral antigens, it has also proved to be an efficient method for the separation of bacterial antigens in studies of polyclonal T cell responses to Bordetella pertussis antigens.

Heterogeneity in the recognition of the simian immunodeficiency virus envelope glycoprotein by CD4+ T cell clones from immunized macaques.

CD4+ T cell recognition of the simian immunodeficiency virus (SIV) surface envelope (env) glycoprotein was examined by using a panel of 10 T cell lines and 4 T cell clones derived from 10 individual macaques immunized with inactivated SIV or recombinant SIV env proteins. The results demonstrated that CD4+ T cells from each animal recognized between 1 and 7 peptides in 4 distinct regions of the protein including both variable and conserved domains. MLR of PBMC from selected macaques together with RFLP analysis by using the HLA DR beta probes suggested that animals of distinct MHC class II haplotypes can recognize identical peptides. These T cell epitopes within conserved regions of the envelope protein, together with identified linear B cell epitopes recognized by neutralizing antibodies, may be relevant in vaccine design.

Services for families of alcoholics: a national survey of Veterans Affairs treatment programs.

Directors of 70 (75% response rate) inpatient and 51 (54% response rate) outpatient alcoholism treatment programs run by the Department of Veterans Affairs (VA) returned questionnaires about patient and program characteristics and the extent of use of family-focused treatment modalities. Referrals to Al-Anon for spouses and to Alateen for teenage children were by far the most frequently provided services. Conjoint couples therapy was the type of direct service provided most frequently. Couples group therapy for outpatient programs and spouse education groups for inpatient programs were the next most frequently used. Family therapy was not used frequently. The majority of programs did not offer services directed at the individual needs of spouses, such as individual or group therapy and educational groups. Finally, services to children of alcoholics were nearly nonexistent being limited to referral of teenage children to Alateen. Marital and family services other than referral to Al-Anon and Alateen and conjoint couple interviews in inpatient programs were provided to fewer patients than those eligible for the services. The extent of family services offered bore little or no relationship to program characteristics (e.g., funding level, staffing pattern) but was related to patient characteristics. Programs with more middle-aged, employed, high school (or better) educated, married veterans made greater use of family-focused modalities. The results suggest the VA needs to develop policy guidelines for the type and intensity of marital and family services their programs should provide with specific determination of what responsibility, if any, VA programs have to the children living with the alcoholic veterans treated in these programs.

Services for the families of alcoholics. A survey of treatment agencies in Massachusetts.

A survey of treatment agencies showed that alcohol misusers' families remain on the periphery of the treatment process. When the spouses or children did receive services, it was usually in the form of individual counseling. Despite their growing popularity, conjoint couples therapy, group couples therapy and family treatment were seldom used.

Inactivation of L-lactate monooxygenase with 2,3-butanedione and phenylglyoxal.

L-Lactate monooxygenase from Mycobacterium phlei is inactivated by reaction either with 2,3-butanedione in borate or in 2,6-lutidine buffer or with phenyglyoxal in 2,6-lutidine buffer. The activation with 2.3 butanedione in borate buffer is irreversible in the presence of excess borate, but essentially complete recovery of activity occurs on exchange of phosphate for borate buffer. In 50 mM borate, inactivation with 2,3-butanedione exhibits saturation kinetics with respect to increasing concentrations of 2,3-butanedione, whereas second-order kinetics for inactivation are seen in 200 mM borate. In 2.6-lutidine buffer, the inactivation is rapid, irreversible on change of buffer, and second order overall. Complete inactivation of the enzyme by phenylglyoxal in 2,6-lutidine buffer occurs on incorporation of 2 equiv of phenylglyoxal per subunit, but only one arginyl residue per subunit is modified. The inactivation is irreversible and second order in phenyglyoxal. There is substantial protection from inactivation in the presence of D-lactate, a competitive inhibitor of the enzyme. It is suggested that an arginyl residue in the active site in L-lactate monooxygenase is involved in the binding of the carboxyl group of substrates to the enzyme. An explanation for the unusual kinetics of inactivation with 2,3-butanedione in borate and with phenylglyoxal in 2,6-lutidine is offered.

Alcohol Control Policies, Alcohol Consumption, and Alcoholism.

This study evaluates the implications of two alcoholism prevention models. The single distribution (log-normal) model posits that the average level of consumption in a society is sufficient to account for the rate of alcoholism; the sociocultural model suggests that variables other than consumption account for alcoholism. Factor analysis and multiple regression were used to assess interstate differences in average alcohol consumption and alcoholism rates. Consumption, controlling for alcoholism rate, was found not wholly to be an economic phenomenon but rather was predicted by urban conditions (a factor measuring unintegrated foreign-born and minority groups and external social control) and two alcohol availability factors. Alcoholism rate was predicted by urban conditions and a social isolation factor, isolated females. Consumption was not a significant predictor of alcoholism in this multivariable analysis. It was concluded that it is an oversimplification to view alcoholism merely as an extension of heavy drinking. Availability is not a unitary dimension and appears, furthermore, to have little potential utility in controlling consumption of alcoholism. Neither of the two availability factors was related to alcoholism; bootlegging appears to be a compensatory mechanism for offsetting low legal availability. The results imply that alcohol control policies and alcoholism prevention need to be directed toward alleviating anomie and social isolation. A variety of efforts toward these ends are suggested: senior citizens programs, minority employment programs, English enhancement training for the foreign-born, etc.

Pain as a joint function of alcohol intake and customary reasons for drinking.

Nonalcoholics who, on a problem drinking inventory, responded "yes" to items reflecting a psychological dependence on alcohol, tended after alcohol ingestion to report a decrease in pain experienced in a cold pressor test. Conversely, those whose responses indicated no psychological dependence on alcohol tended to report pain increases after the consumption of alcohol.

Spicule formation in the calcareous sponge Sycon ciliatum.

The spicule primordium is formed in an intercellular cavity within a group of sclerocytes. This cavity contains organic material which ensheaths the growing spicule but does not appear to determine the nature of the mineral morph (magnesian calcite) or the crystallographic orientation of the spicule. The tip of each growing spicule ray is seated in a 'dense cup' in the cytoplasm of the sclerocyte concerned. Both ends of monaxons are initially inserted each into a dense cup. As rays elongate the sclerocyte membrane around the tip becomes invaginated and forms a system of 'converging spaces' that possibly indicate high secretory activity in that region. Spicule growth involves the displacement and expansion of the organic sheath by the enlarging spicule. Fully formed spicules which are exposed to the mesohyl become surrounded by collagen fibrils. However, these fibrils are in no way concerned with the process of mineral deposition and are never found within the spicule calcite.

Nuclear magnetic resonance studies of sperm whale myoglobin specifically enriched with 13C in the methionine methyl groups.

The Cepsilon methyl group of the 2 methionine residues in sperm whale myoglobin was enriched with respect to 13C. This was accomplished by treatment of the apomyoglobin at pH 4 at room temperature with a 100-fold proportion of 13CH3I to form an intermediate containing enriched S-methylmethionine. Unselective demethylation to regain the apomyoglobin structure was accomplished by treatment at pH 10.5 with 0.5 M dithioerythritol at 37 degrees for 18 h. Reagents were removed at each stage by dialysis against dilute sodium azide solution. Hemin was reincorporated to form the holoprotein in a way that avoided the presence of an excess of the small molecule. After chromatographic purification the enriched myoglobin was obtained in a yield of between 29 and 60%. The composition, absorbance spectrum, circular dichroism spectrum, isoionic point, electrophoretic behavior, and oxygen-binding behavior following reduction were all indistinguishable from those of the virgin protein. NMR measurements were made at 15.1, 25.2, and 67.9 MHz at 27-30 degrees. The two enriched loci are represented by separate resonances that appear slightly downfield of the spectral position of the corresponding resonance in free methionine. The positions of these resonances are sensitive to pH and to the ligand bound at the heme group which is approximately 17 A distant from each methionine Cepsilon. On the basis of two separate types of experiment the downfield resonance was assigned to methionine 55 and the upfield resonance to methionine 131. Part of the observed variations in chemical shift could be treated as arising from pseudocontact interactions but part was ascribed to structural changes communicated to the environment of each methionine residue as a result of changes in heme ligand, pH, or temperature. The linewidths of the methionine Cepsilon resonances are narrowed by increasing temperature according to an Arrhenius energy of activation of nearly 3 kcal. The spin-lattice relaxation times, T1, of the two methionine Cepsilon resonances at the three spectrometer frequencies were interpreted to indicate the existence of rotational motions in each side chain in addition to that about the Sdelta-Cepsilon bond. The results as a whole show that the two methionine side chains undergo continuous variations in environment, and that these variations are controlled by events at a distance within the protein structure. It is suggested that the structural lability serves the function of facilitating conformational variations and adjustments within the heme pocket.

The ultrastructure of Gymnosphaera albida Sassaki, a marine axopodiate protozoon.

Gymnosphaera albida has been found on the sponge Sycon ciliatum in the Menai Straits, North Wales, during the months of May to December. It commonly adopts a sedentary mode of life when cultured, settling with its body in contact with the substratum and its axopodia radiating upwards and outwards all round. At times it floats freely. When sessile it can displace itself, but not by rolling. It is a voracious carnivore. The largest seen had a body size of 510 mum X 320 mum. The body of Gymnosphaera is divided into three zones: a central medulla, a cortex and a superficial reticulated pseudopodial layer. The medulla is finely vacuolated and contains an axoplast at its centre. The cortical cytoplasm contains many nuclei, Golgi bodies, polysomes, mitochondria, osmiophilic globules, lipoid spherules and vacuoles of various kinds, but no zooxanthellae. The superficial reticulated pseuopodial layer contains osmiophilic globules and occasional mitochondria. Axonemes radiate from the axoplast to the axopodia, along which osmiophilic globules are generally in motion. In between the cortex and the reticulated pseudopodial layer there is a narrow, extracytoplasmic capsular wall (Sassaki's line), consisting of a microfibrillar coagulum. The wall is a labile structure, perforating locally to allow the passage of food vacuoles or faeces and vanishing completely in certain conditions. It is evaginated to form a sleeve around the base of each axopodium. The cortex is completely penetrated by a system of clefts, the lumen of which opens here and there into the space containing the capsular wall. The clefts are distinct from the endoplasmic reticulum, cisternae of which are commonly found near the surface of the cytoplasmic tracts. Some of the cortical vacuoles contain organic refractive crystals. The crystals have the shape of crossed rodlets, each rodlet having a thermostable component ensheathing a thermolabile component. Their properties are described. The nuclei are enveloped in a thin layer of cytoplasm, connected by narrow bridges to the adjacent cytoplasmic strands. They generally contain several peripherally arranged nucleoli, each bearing a number of nucleolar organizers. Near the centre of the nucleoplasm there is usually a "central chromatin body". The vacuoles of the medulla are of two kinds, one equipped with a fibrous coat. In the vicinity of an axoneme the coat commonly connects with the microtubules and their cross-bridges. The axoplast has a central "hyalosphere" exhibiting a fibrogranular matrix. No tripartite organelle is present therein. The axoplast shell consists of the proximal ends of the axonemes, each enveloped by a fibrous sheath, the fibres coursing around adjacent axonemes, binding them together. The shell thickness is a constant fraction (1/2.5) of the axoplast diameter. The axonemes consist of bundles of parallel microtubules arranged in transverse section in a pattern of alternating rows of hexagons, the microtubules being joined together by 12.3 nm long cross-bridges...

"Feeling no pain" differential responses to pain by alcoholics and nonalcoholics before and after drinking.

Whisky reduced the level of pain reported by alcoholics but had no effect on that reported by nonalcoholics. The results appear to be based on the joint effects of the alcoholic's expectation that alcohol has an analgesic effect and the physiological cues accompanying alcohol consumption.

The pattern of microtubules in the axonemes of Gymnosphaera albida sassaki: evidence for 13 protofilaments.

The axonemes of Gymnosphaera albida radiate from a central axoplast. Their proximal ends form a shell around the axoplast. Within the shell each axoneme is enveloped by a fibrillar sheath and the microtubules are interconnected by electron-dense linkages, which sometimes appear to be double. In nearly transverse sections the microtubules and their linkages form hexagons of 2 irregular types arranged in alternating rows. The shapes of the hexagons vary from one axoneme to the next. The variation is caused largely by the inclination of the axonemes to the line of sight, but also by distortion occurring during the preparation, observation and photography of the sections. Calculations show that, of a number of likely basic patterns (as would be seen in strictly transverse section), only one is compatible with measurements made on 9 of the axonemes. This involves only one type of hexagon oriented in 2 directions to form a 'parquet-floor' pattern. The hexagon is bilaterally symmetrical and its 6 microtubules all have the same set of angles between their linkages, namely an unpaired angle of 138 degrees 28' and paired angles of 110 degrees 46'. Because these angles are in the ratio of 5:4:4, it is deduced that the microtubules have 13 protofilaments forming their walls. Morphogenetically the lateral growth of the pattern is governed by 2 rules: (1) there must be one, and only one, direction of 2-step zig-zagging of the linkages, and (2) linkages forming opposite sides of a hexagon must be in parallel.