Kirk Sperber

Cytokines in Alzheimer's disease

1. Human astrocytoma cells produced biologically active interleukin-6 when treated with a variety of agents including bacterial lipopolysaccharides, viruses, and interleukin-1. 2. Both human recombinant IL-6 and IL-6 produced by stimulated astrocytes promoted differentiation of cultured neuronal cells and reduced survival time in culture. 3. Interleukin-6 and interleukin-1 stimulated the synthesis of the Alzheimers disease beta-amyloid precursor protein. 4. Cytokines may be involved in stimulation of dystrophic neuritic sprouting, neuronal death, and amyloid deposition noted in the brains of Alzheimers disease patients.

Hydroxychloroquine treatment of patients with human immunodeficiency virus type 1

Hydroxychloroquine (HCQ), an antimalarial agent used to treat patients with autoimmune diseases, has been shown to suppress human immunodeficiency virus type 1 (HIV-1) replication in vitro in T cells and monocytes by inhibiting posttranscriptional modification of the virus. These in vitro observations have been expanded into an in vivo study of HCQ as a potential anti-HIV-1 agent in HIV-1-infected patients. A randomized, double-blind, placebo-controlled clinical trial was conducted in 40 asymptomatic HIV-1-infected patients who had CD4+ counts between 200 and 500 cells/mm3. Patients were randomly assigned to receive either HCQ 800 mg/d or placebo for 8 weeks. Virologic and immunologic parameters, including HIV-1 ribonucleic acid (RNA) via use of polymerase chain reaction, viral culture, antigen and mitogen responses, and proinflammatory cytokine levels were measured at the beginning and end of the study. The amount of recoverable HIV-1 RNA in plasma declined significantly in the HCQ group over the 8-week period (P = 0.022), while it increased in the placebo group. The percentage of CD4+ T cells remained stable in the HCQ-treated group (18.1 +/- 9.2% before treatment vs 18.6 +/- 10.5% after treatment) and fell significantly in the placebo group (21 +/- 7% before treatment vs 19.3 +/- 6.3% after treatment; P = 0.032). However, this was not reflected as a change in absolute CD4+ counts for either group (HCQ, 262.8 +/- 166 cells/mm3 vs 251 +/- 163 cells/mm3; placebo, 312 +/- 121 cells/mm3 vs 321 +/- 124 cells/mm3). Mitogen- and antigen-specific responses remained constant in the HCQ group while T cell proliferative responses to Candida decreased in the placebo group (4.8 +/- 3.6 x 10(3) SI [stimulation index] vs 3.0 +/- 3.0 x 10(3) SI; P = 0.032). Lastly, serum interleukin 6 levels declined in the HCQ group (14.3 +/- 13.5 U/mL vs 12.0 +/- 16.7 U/mL; P = 0.023) but not in the placebo group (11.3 +/- 8.8 U/mL vs 7.0 +/- 11.7 U/mL); this was coincident with a decrease in serum immunoglobulin (Ig)G (2563 +/- 1352 mg/mL vs 2307 +/- 1372 mg/dL; P = 0.032), compared with the placebo group (2733 +/- 1473 mg/dL vs 2709 +/- 1501 mg/dL). No other parameters, including serum p24 and beta-2 microglobulin levels, were altered by HCQ therapy. HCQ thus may be useful in the treatment of patients with HIV-1 infection.

Anti-HIV effects of chloroquine: mechanisms of inhibition and spectrum of activity.

ObjectiveTo investigate the mechanisms and spectrum of the anti-HIV activity of chloroquine. Design and methodsMT-4 cells or peripheral blood mononuclear cells were infected with X4, R5 or R5/X4 HIV-1 strains from clades A–E and HIV-2. The cells were then treated with clinically relevant and achievable chloroquine concentrations (i.e. 0–12.5 μM), so as to determine the EC50. The effects of chloroquine on reverse transcription and integration were tested using a replication-defective reporter HIV-1 construct (pRRL.sin.hPGK.GFP). The effects of the drug on the viral envelope were assessed by syncytium assays and immunoprecipitation, using antibodies to different epitopes of gp120. ResultsIn de-novo infected MT-4 cells, chloroquine selectively inhibited HIV-1 IIIB replication but not pRRL.sin.hPGK.GFP. In chronically HIV-1-infected H9 IIIB cells, chloroquine decreased the infectivity of the newly produced virus and the ability of these cells to form syncytia in co-culture with MT-2 cells. These effects were associated with structural changes in the gp120 glycoprotein, such as a reduction of reactivity with antibodies directed against the glycosylated 2G12 epitope. Although affecting a variable target such as gp120, chloroquine was capable of inhibiting X4, R5 and R5/X4 primary HIV-1 isolates from subtypes A, B, C, D, E and HIV-2. ConclusionAt clinically achievable concentrations chloroquine inhibits HIV-1 post-integrationally by affecting newly produced viral envelope glycoproteins, and the drug has broad-spectrum anti-HIV-1 and HIV-2 activity.

The anti-HIV-1 activity of chloroquine

BACKGROUND there is a dramatic need for drugs with anti-HIV-1 activity that are affordable for resource-poor countries. Chloroquine (CQ) is one such drug. OBJECTIVES to review the data indicating that CQ has anti-HIV-1 activity. RESULTS chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) are endowed with a broad anti-HIV-1 activity inhibiting X4, R5, and X4/R5 stains in lymphocytic and monocytic cells. Interestingly, CQ is capable of inhibiting HIV-1 replication at concentrations within the range reported in plasma of individuals chronically treated with doses of the drug which have well-known and limited toxicity. These effects have been confirmed in vivo in two clinical trials. The principal mechanism of HIV-1 inhibition by CQ seems to be an effect on gp120 on a post-transcriptional level. Because CQ and HCQ appear to have a novel site of action (i.e. post-transcriptional inhibition of gp120), these drugs may be particularly useful in combination with other anti-retroviral agents (e.g. ZDV, ddI and HU). CONCLUSION combining these drugs with other anti-HIV-1 agents, especially HU and ddI, may be an interesting option for the treatment for HIV-1 infected individuals in the developing world.

Comparison of hydroxychloroquine with zidovudine in asymptomatic patients infected with human immunodeficiency virus type 1

Hydroxychloroquine (HCQ), an antimalarial agent used to treat patients with autoimmune diseases, has been shown to suppress human immunodeficiency virus type 1 (HIV-1) replication in T cells and monocytes in vitro by inhibiting posttranscriptional modification of the virus. An initial randomized, placebo-controlled clinical trial conducted in 38 asymptomatic HIV-1-infected patients who had CD4+ counts between 200 and 500 cells/mm3 demonstrated that the amount of recoverable virus declined significantly in the HCQ group compared with the placebo group over the 8-week study period. These preliminary observations were expanded into a second 16-week clinical trial comparing the efficacy of HCQ with that of zidovudine (ZDV) in 72 asymptomatic HIV-1-infected patients with CD4+ counts between 200 and 500 cells/mm3. Patients were randomly assigned to receive either HCQ 800 mg/d (n = 35) or ZDV 500 mg/d (n = 37) for 16 weeks. No adverse reactions to the study medications were observed in either the HCQ or ZDV group. Patients in both groups had reduced levels of recoverable HIV-1 RNA in the plasma, reduced levels of cultured virus, and reduced levels of serum p24 antigen after the 16-week study period. However, no difference was noted in absolute CD4+ counts between the two groups. Interleukin-6 and serum immunoglobulin G levels were significantly reduced in the HCQ group but not in the ZDV group. These findings support the results of the previous clinical trial. Thus HCQ may be potentially useful in the treatment of patients with HIV-1 infection.

Effectiveness of a Specialized Asthma Clinic in Reducing Asthma Morbidity in an Inner-City Minority Population

Asthma is the most common chronic disease of childhood and a leading cause of morbidity in adults. Despite significant advances in medical therapy, asthma morbidity and mortality rates have risen dramatically over the past two decades, especially in minority and socioeconomically disadvantaged populations. Numerous intervention programs have been designed in an attempt to reduce asthma morbidity but few have targeted poor or minority populations. The purpose of this study was to assess whether an outpatient intervention program specifically targeted at a high-minority population in East Harlem, in New York City, was successful in reducing asthma morbidity. A retrospective chart review of 84 patient records was conducted. The patients were divided into two groups, an intervention group (n = 45), who were followed by an asthma specialist (allergist/immunologist), and a nonintervention group, followed by a general internist or pediatrician. Outcome variables including clinic walk-in visits, emergency room visits, and hospitalizations were determined and compared in the pre- and postintervention period in both groups. Patients in the intervention group had reduced total walk-in visits (73 vs. 27, p < 0.001), emergency room visits (30 vs. 5, p < 0.001), and hospitalizations (16 vs. 2, p < 0.001). In contrast, patients in the nonintervention group had no change in total walk-in visits (88 vs. 72), increased emergency visits (7 vs. 22, p < 0.05), and no change in hospitalizations (5 vs. 2), respectively. The outpatient intervention program has been successful in reducing asthma morbidity in the high-risk minority community of East Harlem. Future larger studies are warranted to extend this pilot program to other high-risk minority populations.

Identification of subpopulations of human macrophages through the generation of human macrophage hybridomas

We have generated a series of human macrophage hybridomas by fusing an HGPRT-deficient promonocytic cell line, U937, with macrophages obtained by allowing monocytes to mature into macrophages in teflon bags. The fusions were documented as true hybrids by the acquisition of donor class I molecules, as well as donor derived macrophage surface antigens. The hybridomas represent clonal expansion of individual macrophages, retaining the surface antigen expression and functional capacity of the normal donor cells including cytokine production and stimulation in a mixed lymphocyte reaction. These cell lines differentially express Vmax antigens found on normal macrophages, potentially identifying subpopulations of macrophages. These lines may be useful not only to study normal macrophage function but may be relevant to a variety of disease states where expansion of subpopulations of macrophages identified by Vmax antigens may be important in disease pathogenesis.

Cdc2/Cyclin B1 Interacts with and Modulates Inositol 1,4,5-Trisphosphate Receptor (Type 1) Functions

The resistance of inositol 1,4,5-trisphosphate receptor (IP3R)-deficient cells to multiple forms of apoptosis demonstrates the importance of IP3-gated calcium (Ca2+) release to cellular apoptosis. However, the specific upstream biochemical events leading to IP3-gated Ca2+ release during apoptosis induction are not known. We have shown previously that the cyclin-dependent kinase 1/cyclin B (cdk1/CyB or cdc2/CyB) complex phosphorylates IP3R1 in vitro and in vivo at Ser421 and Thr799. In this study, we show that: 1) the cdc2/CyB complex directly interacts with IP3R1 through Arg391, Arg441, and Arg871; 2) IP3R1 phosphorylation at Thr799 by the cdc2/CyB complex increases IP3 binding; and 3) cdc2/CyB phosphorylation increases IP3-gated Ca2+ release. Taken together, these results demonstrate that cdc2/CyB phosphorylation positively regulates IP3-gated Ca2+ signaling. In addition, identification of a CyB docking site(s) on IP3R1 demonstrates, for the first time, a direct interaction between a cell cycle component and an intracellular calcium release channel. Blocking this phosphorylation event with a specific peptide inhibitor(s) may constitute a new therapy for the treatment of several human immune disorders.

Oral tolerance to protein antigens

Oral tolerance is an active non‐response to antigens delivered via the oral route. Mechanisms governing tolerance induction have been well characterized in mouse. Similar studies in man are lacking, although there is evidence that tolerance can be induced. In disease states, tolerance is altered and this may account for the presence of mucosal inflammation. In food hypersensitivity there is evidence that allergens may be handled differently and this may play a role in disease expression.

Chloroquine exerts an additive in vitro anti-HIV type 1 effect when associated with didanosine and hydroxyurea.

Several groups, including ours, have reported that chloroquine (CQ) or its analog hydroxychloroquine has anti-HIV-1 activity both in vitro and in vivo. We studied in vitro whether the addition of CQ to the combination of hydroxyurea (HU) plus didanosine (ddI) had an additive effect in inhibiting the replication of HIV-1. Therefore both the H-9 T lymphocytic cell line and the U-937 promonocytic cell line as well as primary T cells and monocytes were infected with HIV-1 and then treated with HU at 0.2 mM and ddI at 1 microM and varying concentrations of CQ. Addition of CQ resulted in an additional inhibition of HIV-1 replication, as assessed by reverse transcriptase (RT) activity, with a CQ EC50 of 0.4-0.9 microM for the cell lines and of 0.2-0.9 microM for the primary cells. Similarly, addition of CQ further inhibited HIV-1 replication in U-1 cells stimulated either with LPS or H2O2 and in ACH-2 cells stimulated either with PMA or H2O2, with CQ EC50 values of 0.1 and 1 microM, respectively. Under the experimental conditions used, CQ induced neither toxicity nor apoptosis in the H-9 and U-937 cells. This in vitro additive anti-HIV-1 activity of CQ, in combination with HU + ddI, supports the idea that this triple regimen should be studied in clinical trials. It may become of particular interest to HIV-1-infected individuals from the developing world, in view of the low cost of both CQ and HU.