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Biomedical subjects

A L Goldstein

Publications and source records attributed to A L Goldstein.

At least 19 recordsLinked to original sources
PubMed1992

Thymosin beta 4 (Fx peptide) is a potent regulator of actin polymerization in living cells.

Thymosin beta 4 (beta 4) is a 5-kDa polypeptide originally identified in calf thymus. Although numerous activities have been attributed to beta 4, its physiological role remains elusive. Recently, beta 4 was found to bind actin in human platelet extracts and to inhibit actin polymerization in vitro, raising the possibility that it may be a physiological regulator of actin assembly. To examine this potential function, we have increased the cellular beta 4 concentration by microinjecting synthetic beta 4 into living epithelial cells and fibroblasts. The injection induced a diminution of stress fibers and a dose-dependent depolymerization of actin filaments as indicated by quantitative image analysis of phalloidin binding. Our results show that beta 4 is a potent regulator of actin assembly in living cells.

3T3 Cells↗
PubMed1992

Combination therapy with thymosin alpha 1 potentiates the anti-tumor activity of interleukin-2 with cyclophosphamide in the treatment of the Lewis lung carcinoma in mice.

In this study we have investigated the effects of thymosin alpha 1 (T alpha 1) and interleukin-2 (IL-2), singly or in combination with cyclophosphamide (CY), on tumor growth, survival and cytotoxicity in C57Bl/6NCrlBR mice with Lewis lung carcinoma (3LL). Combined administration of T alpha 1 plus IL-2, after CY treatment, was much more effective than use of each biological response modifier (BRM) alone, and induced complete tumor regression in all of the mice studied. Combination immunotherapy alone without CY only slightly reduced the rate of tumor growth, and these results are in accordance with previous studied which showed that the 3LL carcinoma is resistant to cytokines. Combined chemo-immunotherapy also increased the cytotoxicity of spleen cells and markedly enhanced long-term survival in all treated animals. Depletion of immune cells, using either total-body sub-lethal irradiation (400 rads) or antibodies directed against T-cell (anti-CD4 and CD8) or NK-cell (anti-asialo GM1) populations, abolished the positive response to combination therapy. Histological analysis of the tumors obtained from mice treated with combination chemo-immunotherapy revealed a high number of infiltrating lymphoid cells surrounding a well-circumscribed area of necrosis consisting solely of dead cells. Our studies show that T alpha 1 potentiates IL-2-induced cytotoxic activities in vitro as well in vivo, and that these compounds have a powerful anti-tumor action when associated with chemotherapy.

Animals↗
PubMed1992

Identification of immunoreactive forms of thymosin alpha 1 in serum and supernatants by combining HPLC and RIA.

Thymosin alpha 1 (T alpha 1) is a biologically active peptide, originally isolated from the thymus and currently undergoing clinical trials as an immunomodulator in cancer patients, in individuals with chronic active hepatitis, and as an immunoenhancer of vaccines in immunocompromised individuals. Absorption of rabbit antibody to thymosin alpha 1 with a synthetic C-14 fragment of T alpha 1 results in an antiserum with increased affinity for the amino terminal region of T alpha 1 and the precursor protein prothymosin alpha (ProT alpha). Using HPLC methodologies, the predominant form of immunoreactivity in serum and thymus was T alpha 1 not the precursor. Using this assay we detected a decline in mouse serum T alpha 1 following irradiation but not thymectomy, an observation consistent with the existence of an important radiation sensitive lymphoid source of serum T alpha 1. The secretion of authentic T alpha 1 but not the precursor into culture medium by thymic epithelial cells as well as in mitogen-stimulated peripheral blood lymphocytes was also demonstrated by HPLC/RIA. HPLC analysis by molecular weight sizing columns demonstrated that unlike thymic epithelial cells or peripheral blood lymphocytes, the immunoreactive T alpha 1 (IRT alpha 1) form in the supernatants from tumor cells such as MCF-7 breast carcinoma was of a lower molecular weight than authentic T alpha 1. These studies suggest that the authentic form of T alpha 1 is the major immunoreactive form in normal serum and that it is secreted by the medullary thymic epithelial cells as well as by peripheral blood lymphocytes. An additional immunoreactive form, secreted by tumor cells has also been identified and is the subject of future studies.

Adolescent↗
PubMed1992

Cell-mediated immunity against HGP-30, a group-specific peptide of HIV p17 in individuals infected with the AIDS virus.

HGP-30, the synthetic peptide analogue and active component in an HIV-1 (human immunodeficiency virus, type 1) p 17 core-based experimental vaccine, has previously been shown to induce cytotoxic and helper T-lymphocyte responses. In order to further define the T-helper cell responses which are known to play a role in enhancing the immunological response to foreign antigens, we studied the response of individuals infected with HIV to HGP-30 at various stages of disease progression. We have investigated the proliferative cellular response of peripheral blood mononuclear cells (PBMCs) derived from individuals infected with HIV-1 to HGP-30. We have found a PBMC proliferative response to HGP-30 in 40% of the healthy seroconverted patients, in 35% of the CDC stage III patients and in 18% of the CDC stage IV patients. There was no correlation between the proliferative response to HGP-30 and other antigens such as HIV-like proteins or tetanus toxoid not to CD4 cell count. HLA-DR typing revealed the possible presentation of HGP-30 by several different class II molecules. Since these class II molecules occur frequently in the general population, HGP-30 appears to contain broadly reactive epitopes and thus is not restricted as are many peptide vaccines. Due to its broad reactivity and extreme conservation in many HIV-1 strains. HGP-30 is one of the promising candidates for inclusion as a subunit vaccine against HIV-1.

Acquired Immunodeficiency Syndrome↗
PubMed1992

A phase I study of HGP-30, a 30 amino acid subunit of the human immunodeficiency virus (HIV) p17 synthetic peptide analogue sub-unit vaccine in seronegative subjects.

HGP-30-KLH vaccine in alum at doses of 10, 25, 50, and 100 micrograms/kg administered intramuscularly at weeks 0, 4, and 10 appear well-tolerated clinically. Local pain at the injection site, appears to be the main clinical toxicity. Laboratory parameters are not affected by administration of the vaccine candidate except for perhaps mild urinalysis abnormalities at the highest dose. This vaccine candidate has no apparent immunotoxicity and does not appear to affect lymphocyte populations or T-cell functional studies. Low levels and transient antibodies develop in a minority of subjects early after immunization with the vaccine candidate. These responses were observed in the lowest dose range. Higher doses, and longer follow-up will be needed to confirm this observation. T-cell proliferative responses to KLH and KLH-HGP-30 are consistent and may not be dose dependent, but the proliferative responses are variable and more data need to be accumulated. Preliminary, there appears to be an HGP-30-induced CTL response of HGP-30-coated EBV-transformed autologous B cell lines. This study was approved under an IND for the California Department of Health Services' Food and Drug Branch. They have provided excellent support and regulatory guidelines for this project. Future work will extend and confirm these initial observations.

AIDS Vaccines↗
PubMed1992

Thymosin alpha-1 enhances the fertilizing capacity of human sperm cell: implication in diagnosis and treatment of male infertility.

The effects of synthetic thymosin peptides (T alpha 1 and T beta 4) and their antibodies on the fertilizing capacity of human sperm cells were investigated. T alpha 1, but not the T beta 4, significantly (p < 0.001) increased the human sperm penetration rates in sperm penetration assay (SPA). Antibodies to both T alpha 1 and TB4, which predominantly bound to the acrosomal region of human sperm cell in the indirect immunofluorescence technique (IFT), also significantly (p < 0.001) increased (up to 4.7-fold) the human sperm penetration rates in SPA. The T alpha 1 and antibodies to both T alpha 1 and T beta 4 enhanced spontaneous as well as calcium ionophore-induced acrosome reaction and release of acrosin from the human sperm cells. There was no effect of T alpha 1 and antibodies to T alpha 1 and T beta 4 on percent sperm motility, although they significantly affected various motility characteristics such as velocity, amplitude of lateral head displacement (ALH), and beta frequency--the motility parameters involved in hyperactivation phenomenon of sperm cells. Both T alpha 1 and T beta 4 were detected in the seminal plasma of fertile men, and the levels of T alpha 1 were significantly (p = 0.002) lower in the seminal plasma of infertile men having defective sperm function. These results indicate that the thymosin molecules, especially T alpha 1, may have a role in human sperm capacitation leading to acrosome reaction. These findings also suggest that the T alpha 1 may find clinical applications in the specific diagnosis and treatment of male infertility in humans.

Acrosome↗
PubMed1992

Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA.

We have combined techniques of genetics and histochemistry to identify genes required for the nucleocytoplasmic export of mRNA in the budding yeast Saccharomyces cerevisiae. We adapted in situ hybridization using a digoxigenin-labeled oligo(dT)50 probe to localize poly(A)+ RNA in fixed yeast cells and used yeast strains carrying the rna1-1 mutation to develop an assay. The rna1-1 mutation is the only previously described mutation that causes defects in mRNA export. As visualized with this RNA localization assay, rna1-1 strains accumulated poly(A)+ RNA at the nuclear periphery at the nonpermissive temperature. This was in contrast to the RNA localization pattern of wild-type cells or rna1-1 cells grown at permissive temperature. Wild-type cells showed bright uniform cytoplasmic staining with little detectable RNA in the nuclei. We used this RNA localization assay to screen a bank of temperature-sensitive yeast strains for mutants with inducible defects in mRNA trafficking. Strains identified in this manner are designated RAT mutants for ribonucleic acid trafficking. The rat1-1 allele conferred temperature-sensitive accumulation of poly(A)+ RNA in one to several intranuclear spots that appear to lie at the nuclear periphery. RNA processing was unaffected in rat1-1 strains, except for an inducible defect in trimming the 5' end of the 5.8S rRNA. The wild-type RAT1 gene was cloned by complementation; it encodes an essential 116-kD protein with regions of homology to the protein encoded by SEP1 (also known as DST2, XRN1, KEM1, and RAR5). Sep1p is a nucleic acid binding protein, a 5'----3' exonuclease, and catalyzes DNA strand transfer reactions in vitro. We discuss the possible significance of the Rat1p/Sep1p homology for RNA trafficking. We also discuss the potential of this RNA localization assay to identify genes involved in nuclear structure and RNA metabolism.

Amino Acid Sequence↗
PubMed1992

Specific antibody responses to synthetic peptides of HIV-1 p17 correlate with different stages of HIV-1 infection.

Antibodies were determined against five synthetic peptides (epitopes) of HIV-1 p17 in the sera of an immunologically and clinically well-characterized cohort (N = 292) of HIV-1 seronegative and HIV-1 seropositive high-risk homosexual men, HIV-1 seropositive i.v. drug abusers (IVDA), and AIDS patients. The synthetic peptides, representing the entire HIV-1 p17 protein sequence were: HGP-33 (aa 1-33), HGP-19 (aa 34-52), HGP-35 (aa 51-85), HGP-30 (aa 85-114), and HGP-17 ala (aa 114-131). The presence of one or more peptide-specific antibodies in the sera of all of the HIV-1 p17-positive subjects indicated that all five peptides contain B-cell epitopes. No antibodies were found in the sera of heterosexual controls, HIV-1 seronegative high-risk men, or asymptomatic HIV-1 seropositive but p17 antibody-negative study subjects. Significant differences in antibody recognition profiles to the peptide epitopes were found among the various study groups. A significantly higher proportion of HIV-1 seropositive IVDA had antibodies specific to HGP-17 ala (aa 114-131), HGP-35 (aa 51-85), and HGP-33 (aa 1-33) compared to the HIV-1 p17-positive asymptomatic homosexuals. The epitope-specific antibody responses reflected the clinical status of the HIV-1-infected study subjects, and declined to nondetectable levels as the patient progressed to ARC/AIDS. This decline preceded by several months the reduction in the antibody titer against the intact HIV-1 p17 and p24 proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗
PubMed1991

Low thymosin alpha-1 concentrations in patients chronically infected with the hepatitis B virus.

Serum concentrations of thymosin alpha-1 (TA-1), an immunoregulatory peptide, were assayed in patients with chronic hepatitis B infection and volunteer blood donor controls. Coded samples were tested using a radioimmunoassay. The TA-1 concentrations in HBsAg-positive patients averaged 1555 pg/ml, whereas in the control group, the mean TA-1 in serum was 2159 pg/ml (P less than 0.001). Similar results were found for age- and sex-matched pairs. The TA-1 concentration was not associated with HBe antigen/antibody status or with values of liver-associated enzymes. Analysis of T-cell subsets in nine HBsAg carriers revealed significantly decreased CD4 (helper)-cells, both in absolute numbers and as the percentage of total lymphocytes present. These data suggest that individuals with chronic hepatitis B infection have a relative deficiency of the immunomodulatory peptide TA-1, which may contribute to the characteristics of the resultant T-cell population.

Adult↗
PubMed1991

Complete amino acid sequence analysis of a peptide isolated from the thymus that enhances release of growth hormone and prolactin.

We have previously reported that thymosin fraction 5 (TF5), a partially purified calf thymus preparation, contains a peptide(s) that can enhance the production of GH and/or PRL from rat anterior pituitary cells in vitro. Using reverse phase HPLC, we have now isolated and chemically characterized from TF5 a peptide possessing this activity. This peptide, termed MB-35, is a highly charged basic molecule of 35 amino acid residues and a mol wt of 3756. A computer-assisted search of published protein sequences has revealed that this peptide has a 100% homology with a region of the histone H2A. Biological studies using rat pituitary cells have revealed that MB-35 is active alone or in combination with GH-releasing factor (GRF) or TRH and can increase the production of GH and/or PRL beyond that achievable with GH-releasing factor and TRH alone. The observation that histone H2A, the parent molecule, is without activity is of keen interest, since it suggests that nucleoproteins may have heretofore unknown physiological activities, perhaps related to cell cycle and/or other events associated with DNA activation events.

Amino Acid Sequence↗
PubMed1990

Immune response and epitope mapping of a candidate HIV-1 p17 vaccine HGP30.

A thirty amino acid synthetic peptide (HGP30) representing the conserved region of HIV-1 p17 induced high titer antibodies to the native p17 in rabbits. This immune sera neutralized HIV-1 replication in cell culture and one of the high titer antisera also inhibited CD4-dependent cell fusion. Pepscan analysis with overlapping nonapeptides derived from the sequence of HIV-1 p17 identified the sequence (KE) ALDKIEE (EQ) as the major antibody binding site. Sera of 9% of AIDS patients (7/76) and 18% of HIV-1 seropositive healthy homosexuals (40/223) were positive for HGP30 antibodies. Decline in HIV-1 p17 antibodies has been shown to be related to disease progression in both children and adults, suggesting that HIV-1 p17 antibodies may be protective. Hence, a synthetic HIV-1 p17 peptide, representing the immunodominant epitope, could be useful as a candidate vaccine for immunization of HIV-1 seronegative or seropositive healthy homosexuals.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Thymic hormone modulation of age-induced changes in the induction of graft-versus-host disease by DBA/2J lymphocytes.

Aging induces a number of changes in the immune system, including the involution of the thymus which results in the loss of thymic hormone production and alteration in T cell function. One age-dependent change in immune response is the increasing risk of developing acute or chronic form of graft-versus-host disease (GVHD) following bone marrow transplantation as the age of the recipient increases. A murine model of GVHD that has been extensively studied is one in which injection of C57BL/6 spleen cells into unirradiated B6D2F1 mice results in an acute form of GVHD characterized by cytolytic T lymphocytes (CTL), suppressor cells, runting, and occasionally death. In contrast, injection of DBA/2J spleen cells results in a chronic form of GVHD characterized by a lack of CTL and hyperproduction of immunoglobulin and autoantibodies. This study shows that the GVHD response of DBA/2J spleen cells is dependent on the age of the donor DBA/2J mice. If spleen cells from DBA/2J mice older than 3 months are injected into B6D2F1 recipients, CTL and lack of immunoglobulin production indicative of acute GVHD result. Administration of thymosin fraction 5, a collection of thymic hormones, to DBA/2J mice older than 3 months caused spleen cells from these treated mice to give a GVHD response characteristic of the chronic form of GVHD in B6D2F1 recipients. Thus, thymic hormones were able to modulate the changes in GVHD responses of DBA/2 lymphocytes that occur as the mice age. Preliminary fractionation of TF5 has indicated that there are at least two active thymic peptides present in TF5.

Aging↗
PubMed1990

Changes in circulating levels of neuroendocrine and thymic hormones during aging in rats: a correlation study.

It is well established that during early life the thymus gland and the neuroendocrine system influence each other's maturation. Furthermore, there is a growing body of evidence indicating that the immune and neuroendocrine systems also function as a bidirectional network during adult life. In order to assess possible changes in the thymic-neuroendocrine network during aging, we undertook to measure and correlate the circulating levels of several neuroendocrine and thymic hormones in young (3 month) and old (26 month) male Sprague-Dawley rats. Sequential plasma samples were obtained from chronically cannulated, nonstressed animals every 30 min for 5 h. Two days later rats were killed between 11:30 a.m. and 1:30 p.m. and trunk serum was obtained. All hormones were measured by radioimmunoassay. Growth hormone (GH), prolactin (PRL), thyrotropin (TSH) and corticosterone were measured in plasma, whereas thyroxine (T4), triiodothyronine (T3), thymosin alpha 1 (T alpha 1) and thymosin beta 4 (T beta 4) were determined in trunk serum. The circulating levels of T3, PRL, corticosterone and T beta 4 did not show significant differences between young and old rats, whereas GH, T4, T alpha 1, and thymus weight showed a significant age-related reduction. The anterior pituitary (AP) weight and plasma TSH were significantly higher in old than in young rats. Three pairs of parameters showed highly significant levels of linear correlation: AP weight vs. T alpha 1; thymus weight vs. T4 and T alpha 1 vs. T4 (p less than 0.01, p less than 0.001 and p less than 0.001, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1990

HGP-30, a synthetic analogue of human immunodeficiency virus (HIV) p17, is a target for cytotoxic lymphocytes in HIV-infected individuals.

Evaluation of the immune response of individuals exposed to human immunodeficiency virus (HIV) is an important component of any plan designed to lead toward the development of an AIDS vaccine. Since the levels of antibodies to HIV p17 and the synthetic p17 peptide HGP-30 correlate with stages of progression to AIDS, studies were initiated to determine whether cytotoxic lymphocytes directed toward target cells pulsed with HGP-30 and radioactive chromium were present in seropositive individuals. The significance of such cells in controlling HIV viral infection has recently been enhanced by reports that HIV p17 is on the surface of infected cells and that an inactivated virus vaccine depleted of viral envelope appears to be effective in controlling expression. The selection of HGP-30 as the p17 peptide to be evaluated in early studies is based on the presence of both T-cell and B-cell epitopes as predicted by computer modeling and mouse studies and the demonstration of in vitro neutralization activity by antibodies to the epitope. By using B-lymphoblastoid cells pulsed with HGP-30 and radioactive chromium as autologous targets and mixed leukocyte culture-expanded peripheral blood lymphocytes as effectors, CD8+ cytotoxic T lymphocytes against HGP-30-coated targets were identified in seropositive individuals. In this report we demonstrate that a synthetic p17 epitope can be a target for major histocompatibility complex-restricted cytotoxic T lymphocytes in HIV-infected individuals.

Cells, Cultured↗
PubMed1990

Cellular levels of thymosin immunoreactive peptides are linked to proliferative events: evidence for a nuclear site of action.

Thymosin alpha 1 (T alpha 1), the N-terminal 28-amino acid fragment of prothymosin alpha (ProT alpha), and ProT alpha, although originally isolated from whole thymus extracts, are also present in nonthymic cells and tissues. We used an ELISA with an antibody raised against T alpha 1 to investigate the relationship between intracellular levels of thymosin immunoreactive peptide(s) (TIP) and cell proliferation in a rat small intestinal IEC-6 cell line. Increasing TIP levels were observed during cell proliferation, which decreased when proliferation was halted by cellular contact inhibition. Serum feeding of cells previously rendered quiescent by serum starvation resulted in a significant increase in TIP within 1 hr. Conversely, serum starvation decreased TIP levels within 1 hr. Peak TIP levels appeared after 3 hr of serum incubation, while maximum [3H]thymidine incorporation was noted after 9 hr, suggesting maximum TIP concentrations in the G1 phase of the proliferative cycle. Immunoelectron microscopy demonstrated an association of TIP with condensed nuclear chromatin. These results support a relation of intracellular TIP levels to IEC-6 cell proliferation and also a nuclear site of action. HPLC analysis of cellular homogenates from proliferating IEC-6 cells revealed a peak of immune reactivity that elutes in the position of T alpha 1.

Animals↗
PubMed1990

Peripheral blood mononuclear cell interleukin-2 and interferon-gamma production, cytotoxicity, and antigen-stimulated blastogenesis during experimental rhinovirus infection.

To determine whether rhinovirus infection induced a systemic cellular immune response in humans, specific antigen-stimulated blastogenesis, natural killer cell activity, and mitogen-stimulated production of interleukin-2 and interferon-gamma by peripheral blood mononuclear cells (PBMC) were quantified during experimental rhinovirus infection of normal volunteers. Phytohemagglutinin-stimulated interleukin-2 production by PBMC collected on day 5 after rhinovirus inoculation was increased fourfold compared with production by PBMC collected before rhinovirus challenge (P less than .05); phytohemagglutinin-stimulated interferon-gamma production was doubled (P less than .05). An inverse relationship was observed between the increase in interleukin-2 production and both nasal mucus production (P less than .02) and the number of days virus was cultured from nasal washings (P less than .02). Natural killer cell-mediated cytotoxicity of PBMC collected on day 5 after rhinovirus challenge was also increased (P less than .01) compared with preinfection levels, as was specific antigen-stimulated blastogenesis on day 21 (P less than .05). The extent of blastogenic response correlated directly with both mucus production (P less than .05) and the number of days virus was cultured from nasal washings (P less than .05). These observations are consistent with the hypothesis that rhinovirus infection results in activation of a systemic cellular immune response.

Antibodies, Viral↗
PubMed1989

Synergistic effect of thymosin alpha 1 and alpha beta-interferon on NK activity in tumor-bearing mice.

We have investigated the possibility of thymosin alpha 1 (TH) cooperating with alpha beta-interferon (IFN) in boosting natural killer (NK) activity in tumor-bearing, immunosuppressed mice in vivo. Treatment with a single injection of 30,000 IU of IFN 24 h before testing enhanced NK activity in tumor-bearing mice if the IFN was administered 9 days after tumor inoculation, when the animals have normal NK responsiveness. On the other hand, the same treatment led to lower or no improvement of NK responses if the treatment was given 13 or 17 days after tumor inoculation, at a time when tumor growth causes immunosuppression. However, combination treatment with TH (200 micrograms/kg) for 4 days, followed by IFN was found to restore normal NK cell activity. Selective depletion of antigen-positive cells showed that killer cells stimulated by combination treatment with TH and IFN seem to bear phenotypic characteristics of NK cells. These studies provide the first documentation of a novel combination approach to reconstitution of immunosuppressed tumor-bearing mice using TH and IFN. We hypothesize that TH restores NK boosting activity by IFN by effecting the differentiation/induction of precursor populations of IFN-responsive cells.

Animals↗