Effect of muramyl peptides on mitochondrial respiration.

Muramyl peptides have been shown to exert several biological activities including potentiation of humoral and cell-mediated immunity and stimulation of natural resistance. The mode of action of muramyl peptides has not been elucidated fully and the immunological activities of some derivatives have been associated with toxic effects, including pyrogenicity and inflammatory reactions. Nevertheless, the impact of muramyl peptides on mitochondrial respiration has never been addressed. In this study, the in vitro effects of muramyl peptides on rat liver mitochondria were examined. Toxic muramyl peptides induced a significant decrease in respiratory control ratio versus non-toxic analogues. These results were confirmed by in vivo studies in mice and were extended to mitochondria isolated from spleens. Our data address, for the first time, the effect of muramyl peptides on mitochondrial bioenergetics. Further studies are required to reveal the mechanism of mitochondrial toxicity in relation to the damaging effects of toxic muramyl peptides.

Quantitative study of mitochondria in rat liver. Dry mass, wet mass, volume, and concentration of solids.

The dry mass, wet mass, and volume of mitochondria of normal rat liver were determined, as well as nitrogen content and concentration. A scheme of multiple approaches to these quantities was devised, permitting comparison of values obtained by independent methods. The following basic values are considered highly accurate: Mean dry mass, 13.6 x 10(-14) g; mean wet mass, 51.8 x 10(-14) g; mean volume, 0.43 micro(3); nitrogen content, 1.75 x 10(-14) g The work emphasizes the fact that in mitochondria the quantities, mass, and volume occur in logarithmic-normal distributions.

Eukaryotic initiation factor 5A is a cellular target of the human immunodeficiency virus type 1 Rev activation domain mediating trans-activation.

Expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the presence of the viral trans-activator protein Rev. Rev is localized in the nucleus and binds specifically to the Rev response element (RRE) sequence in viral RNA. Furthermore, the interaction of the Rev activation domain with a cellular cofactor is essential for Rev function in vivo. Using cross-linking experiments and Biospecific Interaction Analysis (BIA) we identify eukaryotic initiation factor 5A (eIF-5A) as a cellular factor binding specifically to the HIV-1 Rev activation domain. Indirect immunofluorescence studies demonstrate that a significant fraction of eIF-5A localizes to the nucleus. We also provide evidence that Rev transactivation is functionally mediated by eIF-5A in Xenopus oocytes. Furthermore, we are able to block Rev function in mammalian cells by antisense inhibition of eIF-5A gene expression. Thus, regulation of HIV-1 gene expression by Rev involves the targeting of RRE-containing RNA to components of the cellular translation initiation complex.

Herpes simplex virus: dry mass.

Dry mass of herpes simplex virus particles was measured by quantitative electron microscopy after isolation by surface spreading and critical-point drying of infected cells. The core weighed about 2 x 10(-16) gram, the empty naked capsid 5 x 10(-16) gram, the full naked capsid 7 x 10(-16) gram, and the enveloped nucleocapsid 13 x 10(-16) gram.

Scanning electron microscopic observations of surface structure of isolated human chromosomes.

Isolated human chromosomes dried by the critical-point method have been assumed to retain their original three-dimensional shape when viewed under a transmission electron microscope. Our scanning electron microscopic study confirms this interpretation and reveals an appearance like that of a skein of yarn. The existence of fiber bridges between chromatid pairs and among chromosomes is demonstrated.

SS-56, a novel cellular target of autoantibody responses in Sjögren syndrome and systemic lupus erythematosus.

Certain autoimmune disorders, including Sjögren syndrome (SS) and systemic lupus erythematosus (SLE), are characterized by autoantibodies against the Ro/SSA and La/SSB cellular antigens. Although the implication of these autoantibodies in disease pathogenesis is still unclear, it is believed that the aberrant responses against autoantigens may extend to other proteins that are not yet well defined. In an attempt to analyze the regulated gene expression in lymphocytes by an HIV-suppressive immunomodulator, we have identified and cloned a novel gene encoding a 56-kDa protein, named SS-56, which is structurally related to the 52-kDa Ro/SSA antigen. The new protein showed primarily perinuclear cytoplasmic localization, and recombinant SS-56 was found to react in ELISA with sera from most patients with SS or SLE. Western blot analysis confirmed the autoantigenic nature of native SS-56 in extracts from HeLa cells. Interestingly, the incidence of antibodies to SS-56 was associated with visceral complications in SLE, and roughly half of the 17 SS or SLE patients with no detectable antibodies to SSA and SSB antigens presented measurable antibodies against recombinant SS-56. Thus, SS-56 represents a new member of the SS family of autoantigens and could become an additional and important diagnostic marker for SS and SLE.

Homogeneously staining regions and double minutes in a human cell line: chromatin organization and DNA content.

Homogeneously staining regions (HSR) and double minutes, two novel chromosome abnormalities, are believed to represent sites of gene amplification. The basic organization of chromatin in an HSR in a human neuroblastoma cell line was shown, by electron microscopy, to be similar to that in a normal chromosome. Double minutes from another human neuroblastoma cell line were found, by dry mass determination, to contain an average of 1.41 X 10(-14) g DNA. This quantity corresponds to 6.38 X 10(3) kilobase DNA per double minute.

Analysis of the antigenic relationship of various derivatives of n-acetyl-muramyl-l-ala-d-isoglutamine (MDP), using anti-MDP antibodies.

Antibodies to N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) were produced in rabbits by injection of MDP conjugated to various carriers [bovine gamma globulin (BGG), methylated BSA or sheep erythrocytes]. The anti-MDP was assayed by a direct enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase linked to MDP-Lys. Various derivatives of MDP were employed in an inhibition of ELISA for analysis of specificity of antibodies and study of the relationship of configuration to biological activity. The results confirmed previous findings that MDP alone is not immunogenic but can act as a hapten when conjugated to carriers. The antibodies were shown to be primarily directed against the muramyl residue. Modifications of this region of MDP yielded derivatives with weak reactivity against anti-MDP, while some changes of other regions had no effect on its antigenicity. Optical isomers of MDP had reduced activity as compared to MDP and polymeric MDP was a strong inhibitor. The structure and function relationship is discussed for some derivatives.

Monoclonal antibodies to the synthetic adjuvant muramyl dipeptide: characterization of the specificity.

Monoclonal antibodies to MDP were prepared by hybridization of NSO myeloma cells with spleen cells of BALB/c mice immunized with MDP conjugated to methyl-BSA. Hybridomas secreting anti-MDP antibodies were selected by the binding activity of their supernates to MDP-A--L using a radioimmunoassay. After cloning in soft agar, the specificities of monoclonal anti-MDP antibodies were assayed by an inhibition of ELISA with various derivatives of MDP. Fine structural analysis of specificity for one such clone (2-4) is reported. This antibody recognizes the N-acetyl-muramic acid (N-Ac-Mur) linked to the dipeptide but not N-Ac-Mur or/and dipeptide alone. The N-Ac group on muramic acid is an important antigenic determinant and the glycopeptide linkage seems to be crucial in presenting the sugar moiety. Conservative substitution of L-Ala (i.e. by L-Ser or L-Val) had no effect on the binding ability to the antibody whereas a radical change, i.e. replacement of L-Ala by L-Pro or N-methyl-L-Ala completely abolished the antigenicity of the molecule. There was no clear correlation between biological activities of various derivatives of MDP and their ability to react with this antibody. Some possible hypotheses explaining this lack of correlation are presented.

MDP(Lysyl)GDP, a nontoxic muramyl dipeptide derivative, inhibits cytokine production by activated macrophages and protects mice from phorbol ester- and oxazolone-induced inflammation.

High levels of pro-inflammatory cytokines and nitric oxide are proposed to orchestrate pathophysiologic mechanism(s) associated with various inflammatory dermatoses. This study examines whether a water soluble 3-O-[N-acetylmuramyl-L-lysyl-D-iso]-2-di-on-glycine [MDP(Lysyl)GDP], a nontoxic and nonpyrogenic derivative of muramyl dipeptide (MDP), can inhibit the in vitro production of inflammatory mediators by lipopolysaccharide- or interferon-gamma-activated macrophages, and whether such an inhibitory effect can translate into in vivo protection of mice from irritant and allergic contact dermatitis. Thioglycollate-elicited peritoneal macrophages cultured in medium alone or in medium supplemented with MDP(Lysyl)GDP (1-100 microg per ml) expressed neither mRNA transcripts for inducible nitric oxide synthase, interleukin-1beta, and tumor necrosis factor-alpha, nor cytokine proteins and nitric oxide activity. Incubation of the cells with either lipopolysaccharide or interferon-gamma for 6 h resulted in a significant induction of inducible nitric oxide synthase, interleukin-1beta, and tumor necrosis factor-alpha mRNA, and the accumulation of high levels of monokines and nitrites in cultures by 24 h. Co-incubation of the macrophages with lipopolysaccharide or interferon-gamma and MDP(Lysyl)GDP (1-100 microg per ml) resulted in a concentration-dependent suppression of the steady-state mRNA transcripts for inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin-1beta, induced by lipopolysaccharide, but not by interferon-gamma. In mouse models of phorbol ester- and oxazolone-induced ear inflammation, topical application of MDP(Lysyl)GDP significantly suppressed ear swelling in a dose-dependent manner. Likewise, oral treatment with MDP(Lysyl)GDP at days -3, -2, and -1 before elicitation with oxazolone also significantly inhibited ear inflammation. Taken together, our findings suggest that MDP(Lysyl)GDP has the potential to be a therapeutic application in the treatment of inflammatory conditions in which overproduction of pro-inflammatory mediators are implicated to play a pathogenic role.

Outcome of cognitive-behavioral and support group brief therapies for depressed, HIV-infected persons.

OBJECTIVE: Although many studies have documented patterns of emotional distress in persons with HIV disease, there have been few controlled evaluations of therapy outcomes with these individuals. This research evaluated the effects of brief cognitive-behavioral or social support group therapy with this population. METHOD: Sixty-eight depressed men with HIV infection were randomly assigned to one of three conditions: eight-session cognitive-behavioral groups, eight-session social support groups, or a comparison condition. Before and after intervention and at 3-month follow-up, all participants were individually assessed by using measures of symptoms of distress as well as substance use and sexual practices. RESULTS: Relative to the comparison group, both the cognitive-behavioral and social support group therapies produced reductions in depression, hostility, and somatization. The social support intervention also produced reductions in overall psychiatric symptoms and tended to reduce maladaptive interpersonal sensitivity, anxiety, and frequency of unprotected receptive anal intercourse, while the cognitive-behavioral intervention resulted in less frequent illicit drug use during the follow-up period. Tests for clinical significance of change particularly underscored benefits of the social support group intervention both at postintervention and at long-term follow-up. CONCLUSIONS: Brief group therapy for depressed persons with HIV infection produced reductions in symptoms of distress. The two forms of therapy resulted in shared and unique improvements in functioning, although social support groups focused on emotional coping presented greater evidence of clinically significant change. As more persons contract HIV infection and live longer with HIV disease, further research is needed to evaluate outcomes of mental health services with these individuals.

AIDS/HIV risk behavior among the chronic mentally ill.

OBJECTIVE: There is growing concern that chronic mentally ill adults living in the community have a high risk for HIV infection. The purpose of this study was to identify risk knowledge, high-risk behaviors, and risk-related encounters of chronic psychiatric outpatients. METHOD: Detailed information on high-risk behaviors and risk-related situations during the past 12 months was collected from 60 outpatients appearing for regular visits at inner-city community mental health clinics. RESULTS: Of the 60 outpatients, 37 (62%) had been sexually active during the past year, and 42% of the men and 19% of the women reported multiple sexual contacts and infrequent use of condoms during intercourse. Assessments of the patients' knowledge of AIDS risks revealed substantial deficits in their practical understanding of AIDS and risk reduction measures. Although use of intravenous drugs was uncommon in this group, many subjects reported histories of 1) trading sex for money, drugs, or a place to stay, 2) coercion to engage in unwanted sex, 3) causal sexual encounters, and 4) sexual activity after use of drugs or intoxicants. Twenty percent of the subjects had met their sexual partners on the streets, in parks, or in other public places. One-third had been treated for sexually transmitted diseases other than AIDS. CONCLUSIONS: These findings underscore the need for AIDS risk assessment, counseling, and prevention programs for the chronic mentally ill.

Enhancement in vivo of the antiinflammatory and antitumor activities of type I interferon by association with the synthetic immunomodulator murabutide.

The therapeutic efficacy of type I interferon (IFN) has been reported to vary considerably in different indications. The use of the cytokine as adjuvant therapy has been suggested to enhance its efficacy and reduce the toxicity frequently associated with long-term and high-dose administration. In this study, we have assessed the activity of type I IFN in the protection against and treatment of acute hepatitis induced in mice by the administration of concanavalin-A (ConA). At the same time, we have evaluated the efficacy of the synthetic immunomodulator murabutide when administered alone or in combination with type I IFN to protect against ConA hepatitis and in the treatment of tumors in MethA sarcoma-bearing mice. Our results demonstrate a prophylactic effect as well therapeutic effects of type I IFN and of murabutide in the inflammation-mediated model of liver damage. The use of combination therapy presented enhanced efficacy in inhibiting the ConA-induced elevation of plasma transaminases. Both compounds were found to suppress IFN-gamma mRNA accumulation in the livers of ConA treated mice. This activity is discussed with respect to the mechanism of action of the two immunomodulators. In addition, the combination of murabutide with type I IFN exhibited synergistic antitumor activity that was clearly seen in the significant regression of MethA tumors and resulted in almost 50 percent tumor-free mice. The potential clinical application of combination therapies using a cytokine and a safe immunomodulator is analyzed in terms of enhancing the cytokine efficacy and extending its use to new indications.

Synergistic effects between recombinant interleukin-2 and the synthetic immunomodulator murabutide: selective enhancement of cytokine release and potentiation of antitumor activity.

The use of interleukin-2 (IL-2) in the treatment of cancer has shown limited efficacy and dose-limiting toxicity. Combination therapy with other cytokines and/or chemotherapeutic agents has been attempted to enhance the antitumor activity and to reduce the effective therapeutic dose of IL-2. We recently showed, in vitro and in vivo, a synergistic activity between the synthetic immunomodulator murabutide, which is in clinical stage of development, and another therapeutic cytokine, interferon-alpha (IFN-alpha). The present study was performed to assess a possible potentiation of the biologic activities of IL-2 by its association with murabutide. Human PBMC stimulated in vitro with IL-2 and murabutide showed synergistic levels of induced mRNA accumulation and protein secretion for IFN-gamma, IL-12, and colony-stimulating factors (CSFs). No such effects were obtained on the induction of most inflammatory cytokines, including IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha). Furthermore, the combined administration of murabutide with IL-2 into Meth-A sarcoma-bearing mice resulted in a very significant tumor inhibition as well as in complete tumor regression in nearly 70% of the treated mice. Under the same conditions, treatment with either compound separately had little or no antitumor effect. These preclinical findings will be pursued by the evaluation of the clinical tolerance and biologic activity of the murabutide/IL-2 combination therapy in cancer patients.

Clinical tolerance and profile of cytokine induction in healthy volunteers following the simultaneous administration of ifn-alpha and the synthetic immunomodulator murabutide.

As the therapeutic use of interferon-alpha (IFN-alpha) is limited by a dose-dependent toxicity and variable efficacy, ways of improving the therapeutic index of the cytokine are being sought. Murabutide (N-acetyl muramyl-L-alanyl-D-glutamine-O-n-butyl-ester) (ISTAC Biotechnology, Lille, France) is a safe synthetic and clinically acceptable immunomodulator that enhances the biologic activities of IFN-alpha in different experimental models. We evaluated in healthy human volunteers tolerance of the coadministration of Murabutide with increasing doses of IFN-alpha. The simultaneous administration of the two drugs was well tolerated without any increased or prohibiting toxicity, and all recipients experienced side effects that were similar to those observed after the administration of IFN-alpha alone. We also profiled the serum levels of cytokines induced following coinjection of the two drugs. We mostly detected an induction of anti-inflammatory cytokines and of human immunodeficiency virus type 1 (HIV-1)-suppressive beta-chemokines, in the absence of release of key proinflammatory cytokines. Therefore, the simultaneous administration of Murabutide and IFN-alpha is well tolerated and does not lead to increased toxicity. In addition, the selectivity in the profile of cytokines and chemokines induced following the coadministration of Murabutide and IFN-alpha points to the potential use of this combination in the treatment of inflammatory diseases and chronic viral infections.

A simplified immunoenzyme antigen binding technique as an approach for immunodiagnosis.

A simple immunoenzyme technique is described in which the enzyme beta-galactosidase of E. coli (Z) is used as a marker. The principle of the test is based on the ability of the cells to bind the antigen. Conditions of the assay using Z both as the antigen and marker are described. As a screening technique, conditions are defined at limited antigen concentrations when binding is not directly proportional to the number of cells present. Hence, drops of blood can be used directly for detection of an immune response without counting the cells. Furthermore, treatment of whole blood with ethanol was shown to (a) increase the binding capacity and (b) allow the storage of specimens for weeks without any loss of activity. With human hydatid fluid (HHF) antigens conjugated to Z as a model, it was possible to detect the immune response of rabbits injected with HHF. The method is simple, requires no sophisticated equipment and can be used in large scale epidemiological surveys.

Detection of intestinal flora-derived bacterial antigen complexes in splenic macrophages of rats.

We studied the presence of bacterial antigens in rat tissues. We produced a monoclonal antibody (MAb 2E9) directed against intestinal flora-derived peptidoglycan-polysaccharide complexes from human and rat feces. With several immunological techniques, the specificity of 2E9 for this bacterial product was demonstrated. Using 2E9 in an immunohistological assay, we were able to show the presence of bacterial products in macrophages in the red pulp of spleens of conventional Lewis rats. However, we found no correlation between the development of the intestinal flora and positive spleen staining with MAb 2E9. The results were confirmed by immunohistology with a previously described MAb 2-4 directed to muramyl dipeptide. Other lymphoid organs did not stain positively with 2E9 and 2-4. Neonatal and young rats showed no staining of the spleen, but positivity could be induced by injecting peptidoglycan-polysaccharide complexes systemically. We conclude that bacterial fragments are present in splenic macrophages of conventional rats.

The effect of photon energy on tests of field uniformity in scintillation cameras: concise communication.

Although Co-57 is generally used for testing the field uniformity of scintillation cameras, the various photon energies of other radionuclides require uniform response throughout the entire range of energies to which a scintillation camera can respond. The use of Co-57, however, may not adequately demonstrate the field response, which may be uniform at 122 keV but not at other energies. Two scintillation camera systems were investigated in this regard by storing field-flood images, obtained at several photon energies, in a minicomputer. The stored data were analyzed using the Kolmogorov-Smirnov test, revealing that field uniformity may change with photon energy. One of the scintillation cameras showed a variation in field response with photon energy, whereas the other camera did not. These results, however, should not be extrapolated to other cameras of the same type. If a particular scintillation camera is to be used routinely with several energies, its performance should be tested with each one to provide assurance that valid information is being obtained. The effects of dynamic uniformity field correction remain to be evaluated.

Clinical comparison of technetium-99m diphosphonate and pyrophosphate in bone scintigraphy: concise communication.

Thirty patients had bone scintigraphy with both Tc-99m pyrophosphate (Tc-PPi) and Tc-99m diphosphonate (Tc-HEDP). The images were given a composite rating for quality and the basis of three sets of criteria, and were also compared for the number of lesions detected by each agent. The two agents provided no difference in scan quality. Nevertheless, in ten of the 30 patients, at least two of the three readers detected with Tc-HEDP lesions that were not seen with Tc-PPi, and in two such cases all three readers considered the Tc-PPi scan normal. In another of these ten, two of three readers felt the Tc-PPi image was norm, whereas all three detected the lesion with Tc-HEDP. The reverse never occurred (P less than 0.01).

Factors associated with severity of depression and high-risk sexual behavior among persons diagnosed with human immunodeficiency virus (HIV) infection.

Whereas some people appear to cope after learning that they have human immunodeficiency virus (HIV) infection, others experience depression and suicidal ideation. In this study, 142 persons with HIV infection were administered the Center for Epidemiological Studies Depression Scale (CES-D). High levels of depression were predicted by lower perceived social support, attributions that health was influenced more by chance, high-risk sexual behavior practices, and greater number of HIV illness symptoms and greater duration of time knowing of one's own positive serostatus. Ongoing high-risk sexual behavior practices were predicted by higher levels of recreational drug use and of depression. These findings highlight the need for improved mental health services for persons with HIV conditions.