In vitro effects of low-level laser irradiation at 660 nm on peripheral blood lymphocytes.

BACKGROUND AND OBJECTIVE: The effects of low-level laser light irradiation are still highly contested, and the mechanisms of its action still unclear. This study was conducted to test the effects of low-level laser irradiation at 660 nm on human lymphocytes and to investigate the possible mechanisms by which these effects are produced. STUDY DESIGN/MATERIALS AND METHODS: Whole blood obtained by phlebotomy was irradiated at 660 nm by using energy fluences between 0 and 5.0 J/cm(2). The lymphocytes were isolated after irradiation of the whole blood. For the control experiment, the lymphocytes were first isolated and then irradiated at the same wavelength and energy fluence for comparison. The proliferation of lymphocytes and the formation of free radicals and lipid peroxides were monitored. Hemoglobin was also irradiated in a cell-free environment to test for the production of lipid peroxides. RESULTS: Lymphocyte proliferation was significantly higher (P<0.05) as expressed by a Stimulation Index in samples irradiated in the presence of whole blood compared with lymphocytes irradiated after isolation from whole blood. Free radical and lipid peroxide production also increased significantly when samples were irradiated in the presence of red blood cells. CONCLUSION: The present study supports the hypothesis that one mechanism for the photobiostimulation effect after irradiation at 660 nm is the reaction of light with hemoglobin, resulting in oxygen radical production.

Mechanisms of adjuvancy: I--Metal oxides as adjuvants.

The exact mechanism of how immune adjuvants function still remains largely unknown, despite their long history of use. This work reports the properties of alum and the related compounds Al(OH)3 or Al2O3. Experiments were performed in rats to determine the relative adjuvancy of silica, talc, ground glass, Al2O3, SnO2, ZrO2, hematite and magnetite. Antibody response and cell-mediated immunity (CMI) to ovalbumin (OVA) were determined and were found to be significantly enhanced by silica and talc. Antibody response to OVA was moderately enhanced by Al2O3, hematite, and magnetite, while CMI to OVA was not affected, SnO2, ZrO2, and ground glass only gave a slight adjuvant effect. The magnitude of adjuvancy appeared to correlate with the magnitude of the inflammatory response produced by each metal oxide and also correlated with their surface area. No correlation could be drawn between the hydrophilicity or hydrophobicity of the metal oxides and the magnitude of their adjuvancy.

Improvement of host response to sepsis by photobiomodulation.

BACKGROUND AND OBJECTIVE: Late sepsis causes immunosuppression and is associated with energy depletion in lymphocytes. Adjuvant treatment with ATP-MgCL2 appears to improve cellular energetics and decrease mortality. Laser irradiation can promote cell proliferation and increase cellular ATP synthesis, which may improve the host immune response in sepsis. The purpose of this study was to determine whether laser irradiation (LI) has a stimulatory effect on the immune response in sepsis using an animal model. STUDY DESIGN/MATERIALS AND METHODS: The cecal ligation and puncture (CLP) rat model was used. Thirty-six SD rats were divided equally among four groups: control (nonoperative), sham operation, CLP treated with laser irradiation, and CLP without laser irradiation. The peritoneal cavity of each animal in CLP/laser group was irradiated immediately after CLP using an Argon-dye laser at a wavelength of 630 nm and at a fluence of 5 J/cm2. Some animals were euthanized 24 hr following CLP and were used to evaluate the immune response (lymphocyte proliferation). In a separate experiment, the survival of septic rats was observed for 60 days. Lymphocytes isolated from normal rat spleens were used to observe for biostimulatory effects in vitro. RESULTS: LI significantly improved ex-vivo lymphocyte proliferation of cells from septic rats (179.7 +/- 17.2 vs. 129.5 +/- 7.8; P < 0.01) and enhanced survival in septic rats (79% vs. 42%; P < 0.001). LI significantly stimulated lymphocyte proliferation in the presence of mitogenic stimuli and enhanced lymphocyte ATP synthesis (P < 0.05). CONCLUSION: LI improves the host immune response and survival rate in sepsis in an animal model. Our studies suggest that LI may be useful as an adjuvant therapy for sepsis.

Adjuvancy effect of different types of silicone gel.

Women with silicone gel-filled breast implants (SBIs) are likely to be at a slightly higher risk of developing an autoimmune-like syndrome. This risk, although small, may be associated with the immunological adjuvancy property of the silicone gel. However, not all silicone gels are chemically formulated exactly the same and their adjuvancy behavior may vary. This study compared, in rats, the adjuvant effect of three different lots of silicone gel using ovalbumin (OVA) as the test antigen. Test bleeds were taken at 21, 48, 62, and 84 days post immunization and the rat sera were analyzed for anti-OVA antibodies by enzyme linked immunosorbent assay (ELISA). A delayed type hypersensitivity (DTH) test was performed on all the treated rats beginning at 14 post-immunization days. The results showed that silicone gel #3 (McGhan lot #S0400488) produced the highest mean anti-OVA antibody titer followed by silicone gel #1 (DC lot #HH019581) and silicone gel #2 (McGhan lot #DP9339). The DTH results showed that rats treated with silicone gel #1 and #3 had a clear positive response, whereas silicone gel #2 caused only a minimal response. These results demonstrate the immunological adjuvancy difference among three types of silicone gel. The chemical composition of each of these silicone gels, that would help explain these results, is yet to be determined.

Induction of type II collagen arthritis in the DA rat using silicone gel as adjuvant.

The Dark Agouti (DA) rat has been shown recently to have a high susceptibility for developing arthritis when challenged with either heterologous or homologous collagen II mixed with mineral oil, or with mineral oil challenge alone. This study determined the arthritogenic potential of silicone gel by either mixing it with bovine collagen II (BII) or by injecting silicone gel alone in DA rats. The incidence of collagen induced arthritis was as follows: PBS group- 0/10, silicone gel group- 4/10, and IFA group- 8/9. Anti-BII antibodies were formed in most of the rats treated with either silicone gel or IFA and these groups of rats showed a positive DTH reaction. The PBS treated rats were negative for both anti-BII antibodies and DTH reaction. The incidence of arthritis formation in rats injected with silicone gel alone was 0/10, while the IFA injected rats showed an incidence of 8/10. Silicone gel taken from a commercial breast implant thus is capable of mediating collagen induced arthritis in the DA rat. However, silicone gel alone does not appear to be arthritogenic.

Induction of type II collagen arthritis in the DA rat using silicone gels and oils as adjuvant.

The relative safety (or otherwise) of silicone gel filled breast implants remains a controversial issue. The Dark Agouti (DA) rat has been shown recently to have a high susceptibility for developing arthritis. This study determined the arthritogenic potential of silicone gel, silicone oil, and the low molecular weight octamethylcyclotetrasiloxane (D4), by either mixing it with bovine collagen II (BII) or by injecting silicone gel alone in DA rats. Three separate experiments were performed using 110 female DA rats with 10 rats per treatment group. The incidence of collagen induced arthritis was as follows: Experiment I (6 micrograms BII)- PBS = 0/10, silicone gel = 4/10, and IFA = 8/9; Experiment II (125 micrograms BII)- PBS = 0/10, silicone gel = 7/10, IFA = 10/10, 1,000 cs silicone oil = 3/10, D4 = 0/10, and 1% D4 in 1,000 cs silicone oil = 1/10; Experiment III (adjuvant alone)-IFA = 8/10, silicone gel = 0/10. Anti-BII antibodies were formed in most of the rats treated with either silicone gel or IFA mixed with BII and these groups of rats showed a positive DTH reaction. The PBS treated rats were negative for both anti-BII antibodies and DTH reaction. Silicone gel taken from a commercial breast implant thus is capable of mediating collagen induced arthritis in the DA rat. However, silicone gel alone does not appear to be arthritogenic.

The effect of molecular weight and gel preparation on humoral adjuvancy of silicone oils and silicone gels.

Silicone gels from commercial breast implants have been shown previously by our laboratory to be potent humoral adjuvants, while the low molecular size 20 centistoke (cs) silicone oil (M.W. 1900) possesses no measurable adjuvant properties. It became necessary to shear the silicone gel during our previous experiments in order to facilitate injection through a syringe and needle, it is conceivable that shearing may reduce the molecular weight of the silicone gel used. This investigation was undertaken to determine whether humoral adjuvancy of silicone oils is dependent on molecular weight and whether the method of shearing the silicone gel affects its adjuvancy. Four Dow Corning 360 Medical silicone oils (100 cs, M.W. approximately 5,000; 350 cs, M.W. approximately 10,000; 1000 cs, M.W. approximately 16,500 and 12,500 cs, M.W. approximately 60,000) and Dow Corning octamethylcyclotetrasiloxane (D4, M.W. 296) were tested for their humoral adjuvancy by immunizing 64 Sprague Dawley rats with 50 micrograms of bovine serum albumin (BSA) mixed with each oil. The rats were periodically bled and the sera were analyzed for anti-BSA antibodies by ELISA. In a separate experiment, three silicone gel preparations with reproducible characteristics were prepared by using a tissue homogenizer and varying the applied shear force. Each of these preparations was tested for its humoral adjuvancy as previously described for silicone oils. Rats immunized with BSA mixed with the highest molecular size silicone oil tested (M.W. approximately 60,000) showed a significant increase in anti-BSA antibodies as compared to the lower molecular size oils. The three silicone gel preparations showed no difference in their adjuvancy effect. Thus, the humoral adjuvancy of silicone oil appears to be dependent on molecular weight. Differential shearing of the silicone gel does not alter its humoral adjuvancy.

The effect of silicone-gel on the immune response.

Silicone materials have been used in medical applications for at least 30 years. Despite this long history of use the question whether silicones can mediate an immunological reaction that may be detrimental to the host remains unanswered. Most studies on the biocompatability of silicones conclude that silicones are chemically stable compounds, which however are often capable of eliciting a benign chronic inflammatory response. Recently, our laboratory has conducted a series of animal experiments aimed at determining the immunological adjuvancy potential of silicone-gel taken from commercial breast implants. Our previous studies have indicated that silicone-gel is a potent humoral (antibody) adjuvant. Our present studies have found that silicone-gel is capable of eliciting auto-antibodies to rat thyroglobulin and bovine collagen II. However this immune response did not produce any histological evidence of thyroiditis or arthritis. Theories to explain why silicone-gel behaves as an adjuvant are discussed along with discussion of the hypothesis on the desirability of replacing silicone-gel with a more hydrophilic material in bioimplants.

The adjuvant effect of silicone-gel on antibody formation in rats.

The extent of immunological adjuvancy of silicone-gel, from mammary implants, up to now, has not been determined definitively. This study compares the immune potentiation effects of silicone-gel with that of Freund's adjuvant, using bovine serum albumin (BSA) as the test antigen in rats. Sixty, 250 gr., male Sprague Dawley rats were divided into six groups: I- phosphate buffered saline (PBS) only, II- silicone oil (Dow Corning Medical Grade 360 liquid silicone), III- 50% silicone-gel (McGhan Medical Corp.- mammary implant) in silicone oil, IV- complete Freund's adjuvant (CFA), V- incomplete Freund's adjuvant (IFA), and VI- 50% silicone oil in IFA. Each adjuvant was mixed or emulsified with an equal volume of 50 micrograms of BSA in 150 microliters of PBS. Each immunization was given intramuscularly in a single injection. Cardiac puncture test bleeds were taken at 12, 22, 40 and 56 days post immunization and the serum anti-BSA-antibody was measured by ELISA. The results indicate that silicone-gel is a potent immunological adjuvant, compared to both CFA and IFA. Silicone oil alone is not as potent as adjuvant and seems to inhibit the immune response when mixed with IFA. There thus appears to be a distinct possibility that silicone-gel may also be able to mediate an auto-immune reaction.

The effect of Nd:YAG laser-induced hyperthermia on local tumor recurrence in experimental rat mammary tumors.

The influence of Nd:YAG laser hyperthermia (45 degrees C for 20 min) on local tumor recurrence followed by CO 2 laser or scalpel excision was studied on 150 F344 female rats that were implanted with R3230AC mammary tumor in the mammary ridge. The development of local tumor recurrence was observed for 39 days postoperatively. All groups undergoing CO 2 laser excision showed a significant reduction in local tumor recurrence (p less than 0.05) compared with the scalpel technique. Animals that underwent CO 2 laser excision and wound sterilization 48 h following laser hyperthermia demonstrated the lowest recurrence when compared with scalpel excision (p less than 0.01). These results indicate that the local thermal effect achieved by laser hyperthermia or laser sterilization plays an important role in the reduction of local tumor recurrence and augments complete local tumor resection.

The generation of antibody in mice to tuftsin: a naturally occurring phagocytosis stimulating tetrapeptide.

Tuftsin (Thr-Lys-Pro-Arg) is a naturally occurring tetrapeptide that stimulates most known functions of the polymorphonuclear leukocyte and macrophage cell lines. We previously reported our unsuccessful attempts to generate antituftsin antibodies by conjugating tuftsin to several carrier proteins and by polymerizing the peptide with glutaraldehyde. To render tuftsin antigenic the following modifications were made to native tuftsin: three glycine residues were added to the N terminus of tuftsin (Gly3-tuf) and cysteine was added to the N terminus (Cys-tuf) and to the C terminus (tuf-Cys). Native tuftsin was covalently conjugated to sheep red blood cells (SRBC). In a separate experiment Balb/c mice primed with SRBC were immunized with 10(7) SRBC peptide conjugate. Native tuftsin and Gly3-tuf were also conjugated to keyhole limpet hemocyanin (KLH). In another experiment KLH and cationized bovine serum albumin (cBSA) were activated with sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (s-SMCC), which was used to control orientation of tuf-Cys and Cys-tuf when conjugated to each carrier protein. All conjugates were administered in complete Freund's adjuvant (CFA) except for cBSA conjugates which were administered in alum. Antibody response was determined by solid phase radioimmunoassay. Results showed that specific antituftsin antibodies were elicited only by Cys-tuf, conjugated to KLH. This study reaffirms that tuftsin is weakly antigenic and confirms the previous work by Gottlieb et al. that antibody to tuftsin can only be elicited when tuftsin is conjugated to the carrier protein KLH in a manner that leaves the peptide carboxyl end free.

The lack of antigenicity of tuftsin: a naturally occurring phagocytosis stimulating tetrapeptide.

Tuftsin (Thr-Lys-Pro-Arg) is a naturally occurring tetrapeptide that stimulates all known functions of the polymorphonuclear leukocyte and macrophage cell lines. Tuftsin is located in the FC 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 (CH3BSA), 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 mammal 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.