Impairment of Non-Specific Immunity in Patients in Persistent Vegetative State

Abstract

In fourtheen patients in persistent vegetative state (PVS) immune responsivenes was investigated. In particular, we studied the relationship between brain lesions following traumatic injury and immune system. In this respect, phagocytosis and killing of Candida albicans by polymorphonuclear cells (PMN) and monocytes were tested. In addition serum levels of Interferon-γ (IFN-γ) were evaluated.

The patients come out fiom PVS by 3–4 month were used as control group.

Data shown a profound impairement of phagocytosis and killing of monocytes and low serum levels of IFNγ when compared with normal values.

Taken together, these findings suggest that brain lesions, may affect non-specific immune response.     

In vivo effects of recombinant-interferon-beta1b treatment on polymorphonuclear cell and monocyte functions and on T-cell-mediated antibacterial activity in patients with relapsing-remitting multiple sclerosis

Treatment with Interferon (IFN)-β has been proposed as a therapeutic approach in multiple sclerosis (MS) patients, mostly in view of its immunomodulating actions. At the same time, evidence has been provided that MS patients exhibit polymorphonuclear cell (PMN) deficits, which can explain the increased susceptibility to infections in these subjects. Here, in 28 patients with relapsingremitting (RR) MS under treatment with recombinant (r)-IFN-β PMN polarization and PMN and monocyte (MO) phagocytosis and killing, as well as T-cell mediated antibacterial activity, were evaluated before treatment and over a period of nine months of treatment.

Our results point out an enhanced rate of polarization (both “spontaneous” or N-formyl-methionyl-leucyl-phenylalanine-induced) in MS patients. After r-IFN-β_1b treatment the polarization rate was further increased. On the contrary, PMN and MO phagocytosis and killing were depressed in comparison to controls and values were further reduced by r-IFN-β_1b treatment.

In patients T-cell mediated antibacterial activity was decreased at TO and dramatically dropped in the course of r-IFN-β_1b therapy.     

Diazepam Inhibits Phagocytosis and Killing Exerted by Polymorphonuclear Cells and Monocytes from Healthy Donors. In Vitro Studies

The effect of a benzodiazepine (BDZ), diazepam on human polymorphonuclear cell (PMN) and monocyte pha gocytosis and killing from healthy volunteers has been evaluated. Diazepam is able to inhibit in vitro both functions exerted by PMN and monocytes at 10^-5 and 10^-6 M concentrations/ 4 × 10⁶ phagocytes. 10^-7 M con centration was not effective in all the instances.

These results are discussed for their possible clinical implications, since previous studies have shown that in patients with phobic disorder there is evidence for reduced phagocytosis and killing capacities.     

Evaluation of the natural immunity in pups inoculated with a modified-live canine parvovirus type 2b (CPV-2b) strain

Three pups 2-4 months old were vaccinated subcutaneously with the modified live canine parvovirus, CPV-2b/29-97 strain. During an observation period of two weeks pups remained clinically health, exhibiting a vigorous post-vaccinal active serological response (haemoagglutinating inhibiting antibody titers for CPV-2 ranging from 1:2560 to 1:5120 at 21 days post inoculation). Phagocytosis and killing of Candida albicans exerted by polymorphonuclear cells and monocytes did not undergo significant modifications 3-6 days post vaccination up to 30 days. Antibacterial activity mediated by peripheral blood lymphocytes (Salmonella typhi was used as a target) was slightly, but not significantly decreased 3 days post vaccination.

Conclusively, in pups the CPV type 2b vaccine seems to be safe as far as natural immune responses are concerned, while its immunogenicity is preserved.     

Relationship between extracellular stimulation of intracellular killing and oxygen-dependent microbicidal systems of monocytes.

Human monocytes require serum components immunoglobulin G, C3/C3b, and B/Bb to exert optimal microbicidal action against ingested microorganisms. The present study was performed to find out whether these factors act by enhancing oxygen-dependent antimicrobial mechanisms. Serum enhanced oxygen consumption and superoxide production by monocytes before phagocytosis, but did not further increase these processes in monocytes that had recently ingested bacteria. Furthermore, serum did not boost iodination during intracellular killing by monocytes. Phorbol myristate acetate, N-formyl-methyonyl-leucyl-phenylalanine, concanavalin A, and concanavalin A-Sephadex all stimulated the conversion of O2 to H2O2 by monocytes, but only concanavalin A augmented intracellular killing. Reactive oxygen intermediates generated by cell-free enzymes (xanthine oxidase or glucose oxidase) in concentrations comparable to those accumulating extracellularly during incubation of monocytes containing bacteria with phorbol myristate acetate did not promote intracellular killing. The presence of catalase during phagocytosis inhibited killing, but had no effect on killing in the postphagocytic state. Monocytes deprived of glucose for 24 h showed markedly impaired O2 consumption, O2- generation, and bacterial killing; all of these effects were rapidly reversed by restoration of glucose. It is concluded that both an intact respiratory burst and extracellular serum factors are necessary for optimal killing of intracellular Staphylococcus aureus by human monocytes. Serum does not appear to act by enhancing the respiratory burst, but rather to have a separate, synergistic role, the biochemical basis of which is unknown.     

In vitro effects of naloxone on T-lymphocyte-dependent antibacterial activity in hepatitis C virus (HCV) infected patients and in inflammatory bowel disease (IBD) patients

Naloxone acts as an opioid antagonist, displacing opioid drugs from cellular receptors. Among opioid substances, β-endorphins are able to bind to several cell receptors, even including those expressed by immune cells. In this respect, evidence has been provided that in the course of viral infections, as well as in patients with ulcerative colitis high levels ofβ-endorphins are detectable. Here, peripheral blood lymphocytes (PBL) from 21 HCV infected patients and 14 patients with IBD, respectively, were incubated with Naloxone and Naloxone + Ca²⁺ in order to evaluate a putative modulation of PBL-mediated antibacterial activity. In fact, previous studies have demonstrated a reduction of this T-cell activity in HCV and IBD patients.

In general terms, the above treatment led to a recovery of the depressed antibacterial activity. In some cases, increase in T lymphocyte function was obtained with Naloxone alone, while in other cases the combination Naloxone + Ca²⁺ gave rise to a restorative effect. Of note, in some instances, lymphocytes were unresponsive to pharmacological modulation.

The overall results suggest that β-endorphins may down modulate T-cell antibacterial response in HCV and in IBD patients by saturating peripheral receptors on immune cells. Therefore, it is likely that Naloxone and/or Naloxone + Ca²⁺ may displace opioid drugs, thus antagonizing their effects.     

Effect of Macrophage Colony-Stimulating Factor (M-CSF) on Mouse Immune Responses in Vivo

We examined the effects of recombinant human M-CSF (rhM-CSF) on mouse macrophages and immune responses in vivo. Intraperitoneal administration of rhM-CSF (20-500 μg/ml) increased Mac-l⁺ cell numbers in the peritoneal cavity. The tumoricidal activities of the macrophages from vehicle-administered (V-MΦ) and from rhM-CSF-administered (M-MΦ) mice were the same as those observed in vitro. However, when activated by lipopolysaccharide (LPS), the tumoricidal activity of M-MΦ was stronger than that of V-MΦ. Intravenous administration of rhM-CSF (500 μ g/gk) increased the number of spleen cells. Flow cytometric analysis showed that administration of rhM-CSF increased Mac-1⁺, B220+ and NK 1.1⁺ cell counts in the spleen. However, CD4⁺ and CD8⁺ cell numbers did not change. Concomitant increases were observed in levels of IL-4 and IL-10 in mouse serum following rhM-CSF administration, but no significant changes were observed in the serum level of IFN-γ.

In experiments involving mouse immune responses, the administration of rhM-CSF reduced the contact sensitivity (CS) reaction against picryl chloride (PC) and augmented IgE production in response to 2,4-dinitrophenyl (DNP), but did not affect the production of either IgM or IgG 1.

These results suggest that administration of rhM-CSF not only activates murine macrophages, but modulates antigen-specific immune responses in vivo.     

Effects of Acetyl-L-Carnitine Oral Administration on Lymphocyte Antibacterial Activity and TNF-α Levels in Patients with Active Pulmonary Tuberculosis. A Randomized Double Blind Versus Placebo Study

Acetyl-L-carnitine (ALC), a drug for the treatment of ageing-related neuroendocrine dysfunctions, was orally administered -2 gm/day for 30 days -to 10 patients with active pulmonary tuberculosis (TBC). Lymphocyte-mediated antibacterial activity and serum levels of tumor necrosis factor (TNF)-U were evaluated before and after treatment, comparing the values with those of 10 TBC patients receiving placebo.

Results show that by day 30, antibacterial activity remained unmodified or increased in ALC-treated subjects, while decreased in the placebo group. No influence of ALC on TNF-U levels was detectable.

These data suggest that the host's immune responses to M. tuberculosis infection can be selectively modulated by drugs acting on the neuroendocrine axis.     

Role of specific antibody in interaction of leptospires with human monocytes and monocyte-derived macrophages

It has previously been shown that human neutrophils ingest and kill nonpathogenic Leptospira biflexa in the absence of serum but that pathogenic Leptospira interrogans is not ingested by neutrophils even in the presence of normal serum. We extended this study by examining the interactions of human monocytes and monocyte-derived macrophages with pathogenic L. interrogans (serovar icterohaemorrhagiae) and evaluating the opsonizing effect of serotype-specific immune serum on the phagocytosis of pathogenic leptospires by monocytes and neutrophils. Leptospires were incubated with monocytes in pellets at 37 degrees C in 5% CO2. No ingestion or killing of pathogenic leptospires occurred when 10% normal serum was used. However, when the pathogenic leptospires were pretreated with serotype-specific immune serum, monocytes or neutrophils in pellets ingested 96% of the organisms and killed 94% of those ingested. Microscopic observations of the interaction confirmed that phagocytosis of the opsonized pathogenic leptospires by monocytes, monocyte-derived macrophages, and neutrophils had occurred. The opsonizing effect of specific antibody may play an important role in the mechanism of host defense against leptospirosis.     

Some immunological aspects of patients with rhinitis in Lebanon

Background: Hitherto immunological determinates in Lebanese patients with rhinitis have not been investigated.

Objective: To identify causative allergens in Lebanese patients with allergic rhinitis and determine possible correlation's among serum allergen specific antibody, polyclonal IgE, IL-4, IL-5 and peripheral eosinophil levels.

Methods: One hundred and thirteen patients with a long lasting history of nasal obstruction, rhinorrhea, sneezing and nasal itching were investigated. Serum allergen specific antibodies using a panel of 10 potential allergens, IL-4 and IL-5 levels were determined by enzyme immunoassays. Polyclonal IgE levels were estimated by an immunochromatographic assay and eosinophil counts by a Coulter STKS counter.

Results: Based on the presence of serum allergen-specific IgE antibodies, 74 patients were considered to have an allergic etiology. Polyclonal IgE levels were elevated in 41 of the 74 allergic rhinitis patients while the other 33 patients had normal serum levels. In the remaining 39 specific IgE antibody-negative patients, 32 had normal, and 7 had elevated, polyclonal IgE levels. IgE specific antibodies to more than one allergen were detected in 59 of 74 patients. The most common causative allergens were mite, Dermatophagoides pteronyssinus, Dpt (83.8%) and Dermatophagoides farinae, Df (78.4%). Analysis of the data indicated that elevated polyclonal IgE levels correlated with the concentration of serum specific IgE antibodies and the number of the detected causative allergens per patient. Fifty-nine of 74 allergic rhinitis patients had elevated IL-4 levels and 44 had elevated IL-5 levels. The number of allergic patients with both elevated IL-4 and IL-5 levels was 24. Finally, only 9 allergic rhinitis patients had peripheral eosinophilia.

Conclusion: Mite Dpt and Df were the most common causative agents of allergic rhinitis in the Lebanese group studied. A prerequisite for Specific Immunotherapy is the identification of the causative allergen. Determinations of polyclonal IgE level and peripheral eosinophil count alone, as an aid to diagnosis are insufficient and may be misleading. On the other hand, determination of all the parameters studied in conjunction appears to be of diagnostic value.     

Effects of Anti-T Cell (Theta) and Anti-B Cell (Beta) Serum on the Immune Response in Mice

Heterologous anti-B cell (anti-beta) serum was prepared in rabbits against the spleen from neonatally thymectomized mice. The anti-beta serum, after absorption with thymus, is cytotoxic for bone marrow, bone marrow-derived cells, fetal liver and peritoneal lymphocytes. The cytotoxicity to the B cell can be absorbed out with bone marrow.

The cytotoxic effects of anti-beta serum on spleen and lymph node cells is compared to that of anti-theta serum. The data suggest that spleen has relatively more B than T cells, while lymph node has relatively more theta-positive cells.

To test the effect of anti-beta and anti-theta serum on the functional activity of lymphoid cells, C57 spleen or thymus was pre-incubated with the antiserum, in the presence of complement, and tested in vivo for graft-vs-host activity or transfer of an adoptive immune response to SRBC.

Treatment with anti-beta serum does not decrease the graft-vs-host activity of thymus or spleen cells. Anti-theta serum does decrease the graft-vs-host activity of both thymus and spleen cells.

Neither anti-beta serum nor anti-theta serum affect the phagocytic activity of peritoneal macrophages.

Both anti-beta serum and anti-theta serum decrease the transfer of an adoptive primary and secondary immune response to SRBC. A combination of anti-theta and anti-beta treated spleen can transfer adoptive immunity. Thymus and bone marrow can reconstitute the immunocompetence of anti-theta or anti-beta treated spleen respectively.

The results suggest that T cells alone can mount a graft-vs-host reaction and that this activity is not affected by anti-beta serum. The transfer of a humoral antibody response, on the other hand, requires functionally active T- and B-cells. This holds true for a primary as well as secondary immune response. Our anti-beta serum does not appear to have any anti-macrophage activity.     

Effects of Bromocriptine Treatment on Immune Responses and 3-Methylcholanthrene-Induced Tumorigenesis in Rats

Seven-week-old male Sprague-Dawley rats were given a single injection of 1.5 mg of 3-methylcholanthrene (3MC) to induce in situ fibrosarcomas. the rats were also treated with the dopamine agonist bromocriptine (BCR) from two days prior to 14 days after 3MC treatment and again for 14 consecutive days beginning at week 5. Tumor incidence was markedly increased and latency decreased in BCR-3MC rats compared to 3MC controls. Natural killer (NK) cell cytotoxicity responses and production of interleukin 2 (IL2) was enhanced at two weeks in rats treated with only BCR. Natural killer cell activity was suppressed at two weeks in rats treated with only 3MC. This effect was reversed by BCR treatment. Rats treated with 3MC and BCR had suppressed NK cell responses and production of IL2 and interferon-γ (IFN) at 12 weeks.

In another study, rats injected with 1, 3 or 5 mg/kg BCR for 14 consecutive days had increased NK cell activity and IL2 production at all doses and increased IFN production at the two high doses. Antibody (IgG) responses to an injected antigen and delayed-type hypersensitivity reactions were not affected by BCR treatment. Animals treated with the two high doses of BCR had decreased serum prolactin (PRL) levels. Serum growth hormone (GH) concentrations were markedly increased in the group treated with 3 mg/kg BCR.

These data suggest that BCR enhances 3MC-induced tumorigenesis. the mechanism of this effect is apparently not mediated by suppression of the immune system since BCR-treated rats had selectively enhanced immune function. Enhancement of immune responses by BCR has not been previously reported.     

Evaluation of nonspecific immunity and plasma levels of interferon-gamma, interleukin-6 and tumor necrosis factor-alpha in preeclampsia.

In this study we evaluated the maternal cell-mediated immune aspects of preeclampsia in terms of phagocytosis and killing of monocytes and polymorphonuclear cells. To evaluate the contribution of cytokines (Cks) in the pathophysiology of preeclampsia, we investigated the plasma levels of interferon-gamma (IFN-gamma), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), respectively, by an enzyme-linked immunosorbent assay. Data showed that phagocytic and killing activities of monocytes were depressed in preeclamptic and normal pregnancies. At the same time, IFN-gamma plasma levels were undectable in both groups. Conversely, we detected significant levels of TNF-alpha in plasma from preeclamptic and normal pregnancies. Moreover, since in three preeclamptic patients the onset of severe preeclampsia was associated with a sharp increased of TNF-alpha plasma levels, we suggest that an increased production of this CK may be implicated in the pathophysiology of preeclampsia.     

Host defense againstMycobacterium-avium complex

Mycobacterium-avium complex (MAC) is an intracellular pathogen and the most common cause of widely disseminated bacterial infection in patients with the acquired immunodeficiency syndrome (AIDS). MAC is infrequently seen in other immunocompromised adults, suggesting that the host defense defect allowing for MAC infection is relatively unique for AIDS. A system was developed for studying the immune response to MAC infection, utilizing MAC isolated from patients with AIDS and monocytes from normal controls and patients with AIDS. Phagocytosis, superoxide anion (SOA) production, and killing were measured. Monocytes from normal controls and AIDS patients were identical with respect to phagocytosis of MAC. In contrast, baseline SOA production was elevated in monocytes from patients compared to normal monocytes and was minimally augmented in response to either phorbol myristate acetate or MAC. Fourteen-day kinetic studies revealed in patients and controls a biphasic pattern with 50–99% killing of AIDS-derived MAC initially, followed always by a rapid out-growth of surviving bacilli. Despite a modest enhancement of MAC killing by normal but not patients' monocytes pretreated with either recombinant interferon- or recombinant tumor necrosis factor-, outgrowth of MAC was always observed in both, typically faster in patients than in controls. Even monocytes in the presence of lymphocytes stimulated with interleukin-2 did not demonstrate enhanced MAC killing. In contrast, high-titered anti-MAC immune serum derived from a patient with polymyositis and disseminated MAC significantly enhanced the killing of MAC by monocytes from both AIDS patients and healthy controls and prevented their out-growth. These findings suggest that the host defense defect allowing for MAC infection appears not to reside in the monocyte and that thein vitro lymphocyte functions examined in this study do not appear to play a major role. What role specific antibody playsin vivo in preventing disseminated MAC is uncertain, but the lack of such antibody may help explain the propensity for AIDS patients to develop systemic infection.     

Monocyte Locomotion in Anergic Chronic Brucellosis Patients: The In Vivo Effect of Ascorbic Acid

In 14 patients suffering from relapsing chronic brucellosis who were anergic to brucella antigens, we have studied peripheral blood monocyte random migration and chemotaxis against non-specific and specific leukoattractants, as well as plasma and monocyte ascorbic acid levels.

We found that all parameters studied, were significantly beneath normal, when compared to normal subjects.

After the oral administration of ascorbic acid at a daily dose of 1gr for 15 conseguetive days, random and directed migration against a non-specific stimulus (casein)returned to normal. Directed migration against disease associated leukoattractants (brucella melitensis and brucella abortus) antigens improved significantly, without reaching normal values.

We concluded that ascorbic acid supplementation might partially restore peripheral, monocyte function and help the monocyte-macrophage system to mount an effective immune response against chronicity of brucella infection.     

The Lack of Antigenicity of Tuftsin: A Naturally Occurring Phagocytosis Stimulating Tetrapsptide

Tuftsin (Thr-Lys-Pro-Arg) is a naturally occurring tetrapeptide that stimulates all known functions of the polymorphomaclear. leukocyte and macrophage cell lines. Tuftsin is located in the F_c region of IgG between the 289 and 292 amino acid sequence of the CH₂ domain. We describe unsuccessful attempts to generate antituftsin antibodies. In separate experiments tuftsin was chemically conjugated to methylated bovine serum albumin (CH₃ BSA), BSA, keyhole limpet hemocyanin (KLH) and purified protein derivative (PPD). Tuftsin was also polymerized with glutaraldehyde. Animals used for immunization were rabbits, roosters, and dogs. All experiments failed to produce antituftsin antibody. Probable reasons for the lack of antigenicity include:

I) Lack of “foreignness” of tuftsin in mammalian species.

II) The small size of the tetrapeptide.

III) Tuftsin may be exerting an adjuvant effect when coupled to foreign antigens and is therefore not recognized by the host immune system.     

Kinetics of Phagocytosis and Intracellular Killing of Staphytococcus aureus and Escherichia coli by Human Monocytes

Tie kinetic patterns of the phagocytosis and intracellular killing of Staphytococcus and Echerichia coli by monocytes were investigated separately to acquire more insight into the total process, i.e. from the ingestion to the death of the micro-organisms. Phagocytosis proved to be dependent on: (1) both the bacteria-to-monocyte ratio and the monocyte concentration; a concentration of at least 5 × 10⁵ monocytes/ml proved necessary for the measurement of ingestion, whereas the rate of ingestion was found to be proportional to the number of extracellular bacteria until a maximum rate is reached, (2) the serum concentration in the incubation medium, which influenced both the rate of phagocytosis and the maximum number of bacteria taken up by one monocyte, and (3) the temperature, the highest rate of phagocytosis being reached at 37–41°C The intracellular killing proved to be dependent on: (1) the number of bacteria ingested; the rate of killing was proportional to the number of ingested bacteria until a maximum rate was reached; (2) the temperature, since a maximum rate of killing is only reached at 37–41°C: at tower and higher temperatures the rate of killing is lower, in the latter case due to inactivation of extracellular stimuli. These separate data on the ingestion and killing processes made it possible to compute the theoretical numbers of extracellular, viable intracellular, and total intracellular bacteria for a model system consisting of 5×10⁶ monocytes, 5×10⁶ bacteria, and 10% serum. These calculated values are in agreement with the experimental data.     

Defects in granulocyte function in various chromosome abnormalities (down's-, edwards'-, cri-du-chat syndrome)

Summary In five infants with autosomal aberrations and diminished resistance to infection (in spite of intact humoral and cellular immune mechanisms) several granulocyte functions (chemotaxis, phagocytosis, intracellular killing and metabolism of killing) were measured. A serum-dependent or a cell-dependent disturbance of phagocytosis of Candida albicans was found in two infants with cat-cry syndrome and one with trisomy 18. In one of these children there was an additional serum dependent defect of the killing of Candida albicans and of Staphylococcus aureus, serum levels of opsonins (IgG, IgM, CH50 and C₃) being within normal range. An infant with trisomy 21 showed, in addition to a cellular defect of chemotaxis, a reduced cellular ability of the killing of Staphylococcus aureus and of Escherichia coli in autologous and AB-pool-serum. Phagocytosis of these bacteria remained normal.     

Critical role for complement receptor 3 (CD11b/CD18), but not for Fc receptors, in killing of Streptococcus pyogenes by neutrophils in human immune serum

During phagocytosis, surface receptors on neutrophils interact with pathogens opsonized with complement factor C3b/iC3b and in some cases with antibodies. In human immune sera antibodies directed against surface-bound M proteins mediated killing of Streptococcus pyogenes by neutrophils. Surprisingly, blocking of the Fc receptors had little effect on the killing. In contrast, inhibition of C3b/iC3b generation, or blocking of the major neutrophil iC3b receptor CD11b/CD18, enabled S. pyogenes to grow efficiently in immune sera. Inhibition of CD11b/CD18, but not of CD32, the major neutrophil signaling Fc receptor, prevented Streptococcus-induced NADPH oxidase-dependent respiratory burst, and blocking of C3b/iC3b formation inhibited Streptococcus-induced activation of Cdc42, a small GTPase critically involved in transmitting pro-inflammatory signals to the cytoskeleton. Consequently, ligation of CD11b/CD18 by bacteria-bound iC3b is necessary for inducing a neutrophil response leading to elimination of S. pyogenes in immune human serum.     

In Vitro Effects of 3'-Azido-3'-Deoxythymidine (AZT) On Normal Human Polymorphonuclear Cell and Monocyte-Macrophage Functional Capacities

The in vitro effects of 3'-azido-3'-deoxythymidine (AZT) (at concentrations of 1, 10 and 100 µM, respectively) on normal human polymorphonuclear cell (PMN) and monocyte-macrophage hnctional capacities were evaluated. Results show that AZT was able to decrease monocyte phagocytosis only, while PMN polarization, phagocytosis and killing were unaffected by drug pretreatment. Quite interestingly, monocyte-derived macrophages maintained their unaltered phagocytic hnction in spite of the presence of AZT in overnight cultures, thus indicating that monocytes are more susceptible than macrophages to the antiproliferative effects of AZT. Since our data indicate that AZT affects normal human monocyte phagocytosis, it is advisable to evaiuate this immune parameter in HIV+ patients administered with this drug.