Darryl M. See

In vitro effects of echinacea and ginseng on natural killer and antibody-dependent cell cytotoxicity in healthy subjects and chronic fatigue syndrome or acquired immunodeficiency syndrome patients

Extracts of Echinacea purpurea and Panax ginseng were evaluated for their capacity to stimulate cellular immune function by peripheral blood mononuclear cells (PBMC) from normal individuals and patients with either the chronic fatigue syndrome or the acquired immunodeficiency syndrome. PBMC isolated on a Ficoll-hypaque density gradient were tested in the presence or absence of varying concentrations of each extract for natural killer (NK) cell activity versus K562 cells and antibody-dependent cellular cytotoxicity (ADCC) against human herpesvirus 6 infected H9 cells. Both echinacea and ginseng, at concentrations > or = 0.1 or 10 micrograms/kg, respectively, significantly enhanced NK-function of all groups. Similarly, the addition of either herb significantly increased ADCC of PBMC from all subject groups. Thus, extracts of Echinacea purpurea and Panax ginseng enhance cellular immune function of PBMC both from normal individuals and patients with depressed cellular immunity.

Cytokine production by adherent and non-adherent mononuclear cells in chronic fatigue syndrome

It has been suggested that cytokines play a role in certain clinical manifestations of chronic fatigue syndrome (CFS). In this study adherent (monocytes) and non-adherent (lymphocytes) mononuclear cells were stimulated in the presence or absence of phytohemagglutinin (PHA) or lipopolysaccharide (LPS), respectively, and supernatants were assayed for IL-6, TNF-alpha, and IL-10 by ELISA. IL-6 was also measured at the mRNA level by polymerase chain reaction. The levels of spontaneously (unstimulated) produced TNF-alpha by non-adherent lymphocytes and spontaneously produced IL-6 by both adherent monocytes and non-adherent lymphocytes were significantly increased as compared to simultaneously studied matched controls. The abnormality of IL-6 was also observed at mRNA level. In contrast, spontaneously produced IL-10 by both adherent and non-adherent cells and by PHA-activated non-adherent cells were decreased. This preliminary study suggests that an aberrant production of cytokines in CFS may play a role in the pathogenesis and in some of the clinical manifestations of CFS.

A Randomized Evaluation of Ethambutol for Prevention of Relapse and Drug Resistance during Treatment of Mycobacterium avium Complex Bacteremia with Clarithromycin-Based Combination Therapy

Patients with AIDS and Mycobacterium avium complex (MAC) bacteremia are at high risk for relapse and emergence of resistant isolates during monotherapy with clarithromycin. Ninety-five AIDS patients with MAC bacteremia received clarithromycin plus clofazimine, with or without ethambutol, in a prospective, multicenter, randomized open-label trial. Of 80 patients with positive baseline cultures, sterilization or a 2 log10 reduction in colony-forming units of MAC in two consecutive blood cultures occurred in 69% of both groups. There were nine relapses in the two-drug arm and three in the three-drug arm. Kaplan-Meier estimates of risk of relapse at 36 weeks were 68% and 12%, respectively (P = .004). All relapse isolates were resistant to clarithromycin. Median time to clarithromycin resistance was 16 weeks with two drugs and 40 weeks with three drugs (P = .004). Ethambutol reduced relapses and emergence of clarithromycin resistance and should be considered an essential component of clarithromycin-based therapies for MAC bacteremia.

Alpha Interferon Treatment of Patients with Chronic Fatigue Syndrome

Thirty patients who fulfilled clinical criteria defined by the CDC for Chronic Fatigue Syndrome were treated with alfa 2a interferon or placebo in a double-blind crossover study. Outcome was evaluated by Natural Killer (NK) cell function, lymphocyte proliferation to mitogens and soluble antigens, CD4/CD8 counts and a 10 item Quality of Life (QOL) survey. Although mean NK function rose from 87.8 +/- 19.6 to 129.3 +/- 20.7 lytic untis (LU; p < .05) with 12 weeks of interferon therapy, there was no significant change in the other immunologic parameters or QOL scores. When the 26 patients who completed the study were stratified according to their baseline NK function and lymphocyte proliferation, 4 groups were identified: 3 patients had normal NK cell function and lymphocyte proliferation when compared to normal, healthy controls, 9 had isolated deficiency in lymphocyte proliferation, 7 had diminished NK function only, and 7 had abnormalities for both parameters. QOL scores were not significantly different for the four groups at baseline. After 12 weeks of interferon therapy, QOL score significantly improved in each of the seven patients with isolated NK cell dysfunction (mean score, 16.3 +/- 7.9) compared to baseline (39.7 +/- 12.1; p < .05). In these patients the mean NK function increased from 35.1 +/- 11.7 to 91.5 +/- 22.7 LU (p < .01). Significant improvement was not recorded for QOL in the other three groups. Thus, therapy with alpha interferon has a significant effect on the QOL of that subgroup of patients with CFS manifesting an isolated decrease in NK function.

INCREASED TUMOR NECROSIS FACTOR ALPHA (TNF-α) AND NATURAL KILLER CELL (NK) FUNCTION USING AN INTEGRATIVE APPROACH IN LATE STAGE CANCERS

Natural products may increase cytotoxic activity of Natural Killer Cells (NK) Tumor Necrosis Factor alpha (TNF-α) while decreasing DNA damage in patients with late-stage cancer. Pilot studies have suggested that a combination of Nutraceuticals can raise NK cell function and TNF-α alpha activity and result in improved clinical outcomes in patients with late stage cancer. The objective of the study is to determine if Nutraceuticals can significantly raise NK function and TNF levels in patients with late stage cancer. After informed consent was obtained, 20 patients with stage IV, end-stage cancer were evaluated (one bladder, five breast, two prostate, one neuroblastoma, two non-small cell lung, three colon, 1 mesothelioma, two lymphoma, one ovarian, one gastric, one osteosarcoma). Transfer Factor Plus (TFP®, 3 tablets 3 times per day), IMUPlus® (non denatured milk whey protein, 40 gm/day); Intravenous (50 to 100 gm/day) and oral (1–2 gm/day) ascorbic acid; Agaricus Blazeii Murill teas (10 gm/day); Immune Modulator Mix (a combination of vitamin, minerals, antioxidants and immune-enhancing natural products); nitrogenated soy extract (high levels of genistein and dadzein) and Andrographis Paniculata (500 mg twice daily) were used. Baseline NK function by standard 4 h 51Cr release assay and TNF alpha and receptor levels were measured by ELISA from resting and phytohemagglutinin (PHA) stimulated adherent and non-adherent Peripheral Blood Mononuclear Cell (PBMC). Total mercaptans and glutathione in plasma were taken and compared to levels measured 6 months later. Complete blood counts and chemistry panels were routinely monitored. As of a mean of 6 months, 16/20 patients were still alive. The 16 survivors had significantly higher NK function than baseline (p<.01 for each) and TNF-α levels in all four cell populations studied (p<.01 for each). Total mercaptans (p<.01) and TNF-α receptor levels were significantly reduced (p<.01). It was also observed that hemoglobin, hematocrit and glutathione levels were significantly elevated. The only toxicity noted was occasional diarrhea and nausea. The quality of life improved for all survivors by SF-36 form evaluation. An aggressive combination of immunoactive Nutraceuticals was effective in significantly increasing NK function, other immune parameters and hemoglobin from PBMC or plasma in patients with late stage cancers. Nutraceutical combinations may be effective in late stage cancers. Clinical outcomes evaluations are ongoing.

The Role of Natural Killer Cells in Viral Infections

Natural killer (NK) cells are important effectors for the lysis of both neoplastic and virus‐infected cells. Lectin‐like receptors on human NK cells, such as NKR‐PIA and CD94, bind to target cell carbohydrate ligands and initiate the lytic process. In addition, P58 and P70 bind to major histocompatibility class I antigens on targets and mediate negative signals. Models using NK cell‐deficient mice have proven useful in elaborating the role of NK cells in the immune defence against multiple viral agents. In addition, studies in humans have suggested a vital role of NK cells in the host defence against human immunodeficiency virus, herpesviruses, hepatitis B and C and other viruses. Several genetic disorders, chronic illnesses and infections have been associated with decreased NK function.

Knowledge-Based Avoidance of Drug-Resistant HIV Mutants

We describe an artificial intelligence (AI) system (CTSHIV) that connects the scientific AIDS literature describing specific HIV drug resistances directly to the Customized Treatment Strategy of a specific HIV patient. Rules in the CTSHIV knowledge base encode knowledge about sequence mutations in the HIV genome that have been found to result in drug resistance in the HIV virus. Rules are applied to the actual HIV sequences of the virus strains infecting the specific patient undergoing clinical treatment in order to infer current drug resistance. A search through mutation sequence space identifies nearby drug resistant mutant strains that might arise. The possible drug treatment regimens currently approved by the US Food and Drug Administration (FDA) are considered and ranked by their estimated ability to avoid identified current and nearby drug resistant mutants. The highest-ranked treatments are recommended to the attending physician. The result is more precise treatment of individual HIV patients, and a decreased tendency to select for drug resistant genes in the global HIV gene pool. The application is currently in use in human clinical trials on HIV patients. Initial results from a small clinical trial are encouraging and further clinical trials are planned. From an AI viewpoint the case study demonstrates the extensibility of knowledge-based systems because it illustrates how existing encoded knowledge can be used to support new applications that were unanticipated when the original knowledge was encoded.

Efficacy of a Polyvalent Inactivated-virus Vaccine in Protecting Mice from Infection with Clinical Strains of Group B Coxsackieviruses

A polyvalent vaccine developed from formalin-inactivated prototype strains of coxsackieviruses group B1-6 was tested for its ability to protect mice from acute infection with clinical strains. Adolescent male CD1 mice received an intraperitoneal injection, repeated once or twice at 8 day intervals, containing 0.3 ml placebo or vaccine. Eight days after the final dose of vaccine, groups of 4 mice were challenged with 1 x 10(4) plaque-forming units of a test strain of group B coxsackievirus, and killed 3 days later. The mean neutralizing antibody titers for the 29 strains tested (4 mice/strain, log2) were 2.2 +/- 0.6, 3.3 +/- 0.9 or 6.4 +/- 1.7 after 1, 2 or 3 doses of vaccine, respectively. In mice receiving 2 or 3 doses of vaccine, titers of virus were significantly lower in the pancreas and/or blood in 7/14 or 14/15 strains, respectively, compared to unvaccinated infected controls. Uninfected, vaccinated mice failed to develop islet cell autoantibodies, histopathological abnormalities in the pancreas, or evidence of viral RNA in the pancreas 12 weeks after 3 doses of vaccine. Thus, prophylaxis with a polyvalent, inactivated-virus vaccine reduced the severity of acute infection with clinical strains of coxsackie group B viruses. A schedule of 3 doses of vaccine was superior to 1 or 2 doses.

THE IN VITRO IMMUNOMODULATORY EFFECTS OF GLYCONUTRIENTS ON PERIPHERAL BLOOD MONONUCLEAR CELLS OF PATIENTS WITH CHRONIC FATIGUE SYNDROME

In humans, eight monosaccharides are required for the synthesis of glycoproteins. Dietary supplements that supply these crucial sugars are known as glyconutrients. A glyconutrient compound was added to Peripheral Blood Mononuclear Cells (PBMC) isolated from normal controls and patients with the Chronic Fatigue Syndrome (CFS), a disease associated with immune dysregulation. The in vitro immunomodulatory effects were investigated. Cell surface expression of the glycoproteins CD5, CD8, and CD11a were significantly lower in patients with CFS compared to normal controls. Addition of glyconutrient homogenate to PBMC from patients with CFS stimulated with phytohemagglutinin significantly increased the expression of each glycoprotein. Furthermore, natural killer (NK) cell function was reduced in CFS patients. The glyconutrient preparation significantly enhanced NK cell activity versus human herpes virus 6 (HHV-6)-infected H9 cells in an 8 h51Cr release assay compared to placebo for PBMC from patients with CFS (p<.01). Finally, apoptosis was significantly higher in patients with CSF. The percentage of apoptotic cells was significantly decreased in PBMC from patients with CFS that had been incubated for 48 h with glyconutrients. Thus, glyconutrients improved abnormal immune parameters in vitro in patients with CFS.

Application of an expert system in the management of HIV-infected patients.

A rule-based expert system, Customized Treatment Strategies for HIV (CTSHIV), which encodes information from the literature on known drug-resistant mutations was developed. Additional rules include ranking and weighting based on antiviral activities, redundant mechanisms of action, overlapping toxicities, relative levels of drug-resistance, and proportion of drug-resistant clones in the HIV quasispecies. Plasma was obtained from HIV-infected patients and the RNA was extracted. Segments of the HIV pol gene encoding the entire protease, reverse transcriptase, and integrase proteins were amplified by reverse transcriptase-polymerase chain reaction (using a total of three primer pairs) and cloned. Sequencing was performed on five clones from each of two patients. When the patients RNA sequencing data were entered into the expert program, and the information was downloaded directly into the CTSHIV program, the five most effective two, three, and four drug regimens coupled with an explanation for their choice were displayed for each patient. Thus, the CTSHIV system couples efficient genetic sequencing with an expert program that recommends regimens based on information in the current medical literature. It may serve as a useful tool in the design of clinical trials and in the management of HIV-infected patients.