Strain differences in the response of German cockroaches (Dictyoptera: Blattellidae) to emulsifiable concentrates. The response of a susceptible and two pyrethroid-resistant field strains of the German cockroach, Blattella germanica, to four emulsifiable concentrates (ECs)--Tempo (cyfluthrin), Demon (cypermethrin), Dursban (chlorpyrifos) LO, and XRM 5184 (chlorpyrifos)--was investigated. Application rates were 0.4, 0.1, 0.5, and 0.5%, respectively. Susceptible cockroaches avoided dried formulations of the pyrethroids Tempo and Demon, but resistant cockroaches did not; avoidance was greater with Demon than with Tempo. Filter papers freshly impregnated (wet) with Demon and Tempo flushed susceptible cockroaches (Demon greater than Tempo). Resistant cockroaches were flushed only by Demon, but less so than susceptible strain cockroaches. No mortality occurred in experiments with the pyrethroids. Dried formulations of Dursban LO and XRM 5184 had little, if any, repellency to nymphs of either the susceptible or resistant strains. Initially (1-3 h), most cockroaches settled on treated papers. Subsequent movement off treated papers was a step in the process leading to knockdown. At 24 h, mortality of susceptible cockroaches was 100% in the experiment with Dursban LO and 95% in the experiment with XRM 5184. Highest mortality in a resistant strain (40%) was in the contact experiment with Dursban LO. In contact repellency and flushing experiments, mortality was higher with Dursban LO than with XRM 5184. Neither chlorpyrifos formulation flushed cockroaches effectively. Mortality in flushing experiments was less than in the contact repellency experiments.


Improving bond strength through acid etching of dentin and bonding to wet dentin surfaces. A bonding system using moisture on the tooth surface can be an enormous benefit as obtaining dentin dryness in the mouth is nearly impossible. The author describes a system that bonds to both wet and dry surfaces.


Recognizing and reporting victims of domestic violence. Domestic violence can be defined as any violent behavior directed against an individual within the home or family. It occurs in all segments of society and is not limited to a single ethnic or socioeconomic group. Since most intentional injuries occur in the head and neck area, the dentist might be the first to treat the domestic violence victim.


Computer viruses: a valid threat for the health care professional? The probability of a virus infecting a health care professional's computer is minimal if prevention is used. However, all office personnel who access the computer must be educated with what to look for, what to do and what not to do when a computer virus is suspect.


Principles of ethics and code of professional conduct with official advisory opinions revised to May 1992. The ethical statements which have historically been subscribed to by the dental profession have had the benefit of the patient as their primary goal. Recognition of this goal, and of the education and training of a dentist, has resulted in society affording to the profession the privilege and obligation of self-government. The Association calls upon members of the profession to be caring and fair in their contact with patients. Although the structure of society may change, the overriding obligation of the dentist will always remain the duty to provide quality care in a competent and timely manner. All members must protect and preserve the high standards of oral health care provided to the public by the profession. They must strive to improve the care delivered--through education, training, research and, most of all, adherence to a stringent code of ethics, structured to meet the needs of the patient.


Enhancement of polymorphonuclear cell phagocytosis by lipid A-activated monocytes via cell-to-cell contact. A possible role for membrane-associated cytokines. Previous findings have shown that lipopolysaccharide (LPS)-activated human monocytes express cytokines (CKs) on their membrane. Furthermore, those associated to membrane products such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 have been demonstrated to exert many biological activities. In this paper, evidence is provided that human polymorphonuclear cells (PMN) exhibited an increased phagocytic capacity following incubation with either lipid A (LA)-activated autologous monocytes or supernatants recovered from LA-stimulated mononuclear cell cultures. In order to investigate the possible role of monocyte membrane-associated TNF-alpha, IL-1 alpha and IL-1 beta in the modulation of PMN activity, in a separate series of experiments LA-activated monocytes or LA-activated supernatants were pretreated with anti-recombinant human (Rhu) TNF alpha, anti-Rhu IL-1 alpha and anti-Rhu IL-1 beta monoclonal antibodies (MoAbs), respectively. Such an approach gave rise to an abrogation of monocyte-mediated triggering effect on PMN functional capacity. Taken together, these data suggest that activated monocytes can upregulate PMN phagocytosis by a cell-to-cell contact mechanism, likely related to membrane-associated CKs.


Immunological consequences of zidovudine treatment in control and morphine or methadone treated mice. The effects of acute and chronic zidovudine (AZT) administration on immunologic test responses of mice were studied. The effects of AZT administration combined with morphine or methadone treatment, were also studied separately comparing the effects of each drug. We noted that AZT-treatment did not modify the T-lymphocyte subsets (L3T4/LyT2 rate), whereas morphine-treatment and AZT plus morphine treatment decreased the percentage of T helper cells. Acute and chronic AZT-treatment increased Natural Killer cell (NK) activity and also recovered the decreased NK cell activity produced by morphine-treatment. AZT-treatment, morphine-treatment, AZT plus morphine treatment and AZT plus methadone treatment strongly depressed the phagocytic physiological activity of Polymorphonuclear leukocytes (PMNs). Another evidence of immunologic responsiveness against AZT was the reduction of the mitogenic and antigenic response of lymphocytes. These results suggest a negative role of AZT-treatment especially on phagocytic activity and confirms a depressive effect of morphine-treatment on several immune functions studied. Furthermore, there is no indication of additive or synergistic toxic effects of AZT, morphine and methadone on the immune functions above that seen with each of these drugs when tested alone.


Effect of a traditional Chinese medicine, bu-zhong-yi-qi-tang (Japanese name: Hochu-ekki-to) on the protection against Listeria monocytogenes infection in mice. Effects of Bu-Zhong-Yi-Qi-Tang (Japanese name: Hochu-ekki-to) on the resistance against Listeria monocytogenes were observed in ICR mice orally administered this medicine daily for 10 days. Survival rates were increased by the pretreatment in mice inoculated i.v. with bacteria 1 day after the last administration and in mice inoculated i.p. 4 days after the last administration. After an i.v. inoculation of L. monocytogenes, the numbers of bacteria in the spleen and liver increased gradually to kill mice by day 5 in untreated group but the bacterial numbers increased slightly by day 3 and decreased from day 3 to day 8 in Hochu-ekki-to pretreated group. After an i.p. inoculation, the number of bacteria in the peritoneal cavity decreased very rapidly within 6h in Hochu-ekki-to treated group compared to that in untreated group. After the administration, number of polymorphonuclear cells increased in the peripheral blood, peritoneal cavity and spleen. In treated mice, macrophages increased in number in the peritoneal cavity and the spleen but decreased in the peripheral blood. Peritoneal macrophages from treated mice showed an enhanced activity to kill L. monocytogenes in vitro within 60 min after ingestion of bacteria. Hochu-ekki-to may augment the host defense against L. monocytogenes through the activation of macrophage series during an early phase of infection.


Augmentation of mitogen-induced interleukin 2 production by oral administration of polysaccharide SPR-901. The effects of oral administration of antitumor polysaccharide SPR-901 (RBS), an alpha-1,3 branched alpha-1,6 glucan, on concanavalin A (Con A)-induced interleukin 2 (IL-2) production of splenocytes were studied. The augmentation effect on Con A-induced IL-2 production was evident when more than 30 mg/kg of SPR-901 was administered orally to mice. On the other hand, oral administration of B512 dextran, an analogous alpha-1,6 glucan, did not show any augmentation effects on IL-2 production. The augmentation effect of SPR-901 on IL-2 production seemed to be mediated mainly by macrophages stimulated with SPR-901.


Anesthetic agents induce human mononuclear leucocytes to release cytokines. Studies were carried out on the ability of some anesthetic agents (Propofol, Dormicum, Ketalar and Penthotal) to induce the release of cytokines by human monocytes and lymphocytes in vitro. All anesthetic agents tested at hematic concentrations reached during anesthetic administration cause an increase in the production of Tumor necrosis factor (TNF) from human monocytes; the increase is 4-5 times greater than controls. The greatest Interleukin -1 alpha (IL-1 alpha) production increase was induced by Propofol. The release of Interleukin -6 (IL-6) is notably increased by Ketalar (about 10 times greater than controls). In the presence of different anesthetic agents, human lymphocytes release Interleukin -4 (IL-4) and Interferon gamma- (IFN-gamma). Penthotal and Ketalar increase IL-4 production which appears quite high compared to that obtained with Con A used as standard challenge. Propofol induce IL-4 release which is about the same as that seen with Con A. IFN-gamma is released in high quantities by lymphocytes treated with Propofol. Dormicum, Ketalar and Penthotal induce non-significant increase of IFN-gamma release. The results concern the choice of anesthetic, in relation to its action on host immune response. This aspect is particularly interesting in immunocompromised host.


Serum tumor necrosis factor activity in inflammatory bowel disease. Serum tumor necrosis factor (TNF) levels in 33 patients with inflammatory bowel disease (IBD) were measured by using a sensitive enzyme immunoassay. Four of five Crohn's diseases (CD) and nine of twenty eight ulcerative colitis (UC) had elevated levels of serum TNF. In active CD or UC, a greater fraction of patients studied had significantly increased serum TNF levels (3/3 for CD and 8/11 for UC). Production of TNF by peripheral blood monocytes when stimulated by lipopolysaccharide was also increased in these patients and correlated with their serum TNF levels. These results suggest that TNF may have some pathoetiological meaning in IBD.


Nicotine enhances interleukin production of rat splenic T lymphocytes. Very little is known regarding the effects of nicotine, the most pharmacologically active component of tobacco products, on T lymphocyte activity or interleukin production. Therefore, rats were implanted subcutaneously with osmotic mini-pumps containing either physiological saline, nicotine (1.5 mg/kg/day) or a high dose of nicotine (4.5 mg/kg/day) for a period of 14 days. The ability of the splenic T lymphocytes to respond to the polyclonal T lymphocyte mitogens, Concanavalin A (ConA) or phytohemagglutinin (PHA), and the ability of mitogen stimulated splenic T lymphocytes to produce interleukin 2 (IL2) were determined. Treatment with nicotine suppressed, in a dose dependent fashion, the ability of splenic T lymphocytes to respond to mitogen, but dramatically enhanced the ability of mitogen stimulated lymphocytes to generate IL2.


Effects of famotidine on various immunological parameters in patients with rheumatic diseases: decreased lymphocyte DR expression by famotidine. We examined the effect of famotidine, a histamine-type 2 receptor antagonist, on the immunocompetent cells. The number of DR(+) cells were significantly decreased in patients with systemic lupus erythematosus (P less than 0.05) by parenteral administration of famotidine (40 mg/days for 4 weeks). However, total lymphocyte number and monocyte number did not change. Immunoglobulin levels of patients with rheumatoid arthritis and normal male did not change. Furthermore, phytohemagglutinin induced lymphocyte proliferation was increased by addition of famotidine (10 ng/ml). Nonetheless, famotidine did not have mitogenic function itself to lymphocyte and did not affect IL-2 production.


Immunotoxic effects of mercuric compounds on human lymphocytes and monocytes. I. Suppression of T-cell activation. Considerable attention has been directed at defining the health deficits associated with exposure to mercurial compounds. While numerous studies have been conducted, the findings have been somewhat contradictory and have led to a confused understanding of the immunotoxicology of mercury. It is becoming clear, however, that the immunotoxic effects of heavy metals in general, and mercury in particular, are dependent upon the assays and source of cells. The major goal of our study was to assess whether low level mercury exposure modulates human T-cell function. Following treatment of T-cells with HgCl2 (0-1000 ng) and MeHgCl (0-100 ng), their activation by mitogens was evaluated. Both forms of mercury caused a dose dependent reduction in T cell proliferation, however, the effect was dependent upon the presence of monocytes. Moreover, in the absence of monocytes, HgCl2 enhance PMA induced T-cell proliferation. MeHgCl was approximately 5-10 times more potent than HgCl2. Mercury also inhibited the ability of these cells to synthesize and secrete IL-1. Analysis of the expression of activation markers on the cell surface indicated that one of the earliest markers of lymphocyte activation, CD69, was not effected by mercury. In comparison, T-cell expression of IL-2R and the transferrin receptor was impaired. Of particular interest, cells activated by mitogen for 24 hr became refractory to the immunotoxic effects of mercury. The results of this investigation clearly show that mercury-containing compounds are immunomodulatory; moreover, the decrease in T-cell function following exposure to mercury indicates that this metal is immunotoxic at very low exposure levels.


Immunotoxic effects of mercuric compounds on human lymphocytes and monocytes. II. Alterations in cell viability. The major goal of this investigation was to examine the cytotoxic properties of both HgCl2 and MeHgCl, in terms of their ability to alter human T-cell and monocyte viability. Following treatment with HgCl2 (0-20 micrograms/ml) or MeHgCl (0-2 micrograms/ml), there was minimal reduction in lymphocyte viability at 1-4 hr. However, after exposure to mercury for 24 hr, cell death was apparent. In comparison, monocytes exhibited significant loss of viability during the early exposure periods. MeHgCl was approximately 5-10 times more potent than HgCl2. Other indicators of cell death were also determined. Measurement of the energy charge ratio indicated profound changes in cellular energy conservation. Electron microscopic analysis of cells treated with mercury revealed early nuclear alterations characterized by hyperchromaticity, nuclear fragmentation and condensation of nucleoplasm. In concert with these nuclear changes, there was destruction of cytoplasmic organelles with loss of membrane integrity. Studies of phospholipid synthesis by mercury treated cells confirmed that there were alterations in membrane structure. Thus, there was a decrease in total phosphatide synthesis by treated cells. Moreover, monocyte phospholipid synthesis appeared to be more sensitive to the presence of mercury then lymphocytes. Finally, both forms of mercury caused a rapid and sustained elevation in the intracellular levels of Ca++. These morphological and biochemical changes are consistent with the notion that mercury initiates cytotoxic changes associated with programmed cell death.


Effect of human IgG1 peptides on the antigen-specific antibody response of mice in vivo. The effect of synthetic peptides--corresponding to the amino acid sequences 289-301 (Y48) and 293-301 (Y91) within the CH-2 domain in the human IgG1 was studied on the oxazolone-specific primary and secondary antibody response isotype distribution and on the sheep erythrocyte (SRBC)-specific primary IgM response. High responder (Balb/c) and low responder (C57Bl/6) mice to oxazolone hapten were treated intraperitoneally with various doses of peptides simultaneously with the first and second contact sensitization. The relative levels of oxazolone-specific IgM, IgG3, IgG1, IgG2a and IgG2b antibodies were determined by a solid phase radioimmunoassay. Y48 and Y91 peptides in a dose range of 10(-5) - 10(-8) M/animal enhanced the oxazolone-specific antibody response. This effect was more striking under suboptimal conditions: using smaller antigen dose for sensitization, cyclophosphamide pretreatment or using genetically low responder mice. SRBC-specific primary IgM response was enhanced by Y91 peptide, Y48 was ineffective.


Chemotactic activities of peripheral blood polymorphonuclear leukocytes and peritoneal exudate polymorphonuclear leukocytes in MRL mice. Chemotactic responsiveness to fMet-Leu-Phe in concentrations of 10(-8) to 10(-4) M in the Boyden chamber was compared between peritoneal exudate cells (PEC) and polymorphonuclear leukocytes (PMN) isolated from peripheral blood, between MRL/Mp-+/+ (MRL-+/+) and MRL/Mp-lpr/lpr (MRL-lpr/lpr) mice, and between young (6-9 week old) and aged (16-24 week old) mice. Chemotactic responsiveness of PEC did not differ between MRL-+/+ and MRL-lpr/lpr, and young and aged mice. While, PMN showed greater chemotaxis in aged MRL-+/+ mice than that in aged MRL-lpr/lpr mice. These results suggest that chemotactic responsiveness of PMN differ from that of PEC which is assumed to be preactivated by an inflammatory agent injected into the peritoneal cavity to elicit cells. Less responsiveness of PMN to the bacterial origin peptide might relate to the autoimmune disease of this murine model.


Expression of c-fos and c-myc oncogenes in the P388D1 murine macrophage line treated by immunomodulators: absence of direct correlation with DNA synthesis. Complex patterns of metabolic and functional characteristics are induced in macrophages by biological response modifiers. The study of the early events resulting from the transduction of immunomodulatory signals could be an approach for a better understanding of this activation process. The transcription of c-fos and c-myc genes has been shown to be rapidly modified in many cells responding to various signals. Since murine peritoneal macrophages are a rather heterogeneous population we chose to investigate the c-fos and c-myc modulation in the P388D1 murine macrophage line. Owing to the frequent implication of the c-myc gene in the tumorigenicity of hematopoietic cells we first demonstrated the normal c-myc status in this cell line by Southern analysis. The modulation of the c-fos and c-myc expression has been studied by Northern analysis, 15, 30 and 60 minutes after treatment of the P388D1 cells by the phorbol ester (TPA), the Calcium ionophore A 23187 (Ca2+I), the N-acetyl muramyldipeptide (MDP) or the macrophage Colony Stimulating Factor (CSF-1). The mitogenic activity of these compounds, as evaluated by [3H] thymidine incorporation, has been measured either after a 30 minute or a 24 hour treatment. An early increase in c-fos expression always preceded a c-myc augmentation. The highest modulation of c-fos and c-myc was observed with TPA. Ca2+I and TPA presented a low mitogenic effect if compared to CSF-1. MDP did not change DNA synthesis even after 24 hours. Therefore, in the present study on the P388D1 macrophage cell line, no direct correlation could be evidenced between the mitogenic effect and the modulation of c-fos and c-myc induced by these immunomodifiers. Investigations are in progress in order to evaluate the role of these proto-oncogenes on terminal differentiation induced by immunomodulators in this cell line.


Differential effects of morphine and naltrexone on the antibody response in various mouse strains. Morphine treatment has been shown to suppress several immunologic parameters. In this study, we examined the effects of morphine pellet implantation in vivo on the primary antibody response measured in vitro in various mouse strains. Effects of mouse strain and sex on morphine-induced suppression of the plaque-forming cell response, as well as spleen weight and mortality were determined. Morphine suppressed the primary antibody response in C3HeB/FeJ, C3H/HeJ and C57Bl/6 mice, while Balb/cByJ and the mu-receptor-deficient strain CxBk/ByJ mice were not affected. There was no difference in the response to morphine between male and female C3HeB/FeJ mice. Naltrexone reversed the morphine-induced suppression in the C3H strains, but not in C57Bl/6 mice. In addition, naltrexone caused significant mortality in Balb/cByJ mice. Spleen weight was decreased by morphine treatment in all the strains, but only the C3H strains were sensitive to the lethal effects of morphine. Thus, immune suppression did not correlate with splenic atrophy or mortality. The strain differences in response to chronic morphine and naltrexone treatment suggest that morphine may be acting through both opioid and non-classical opioid (e.g., not blocked by naltrexone) mechanisms.


Continuous infusion of endotoxin depresses splenic blastogenesis. Continuous infusion of a sublethal dose of bacterial endotoxin into rats via an implanted osmotic pump markedly affected the blastogenic responsiveness of spleen cells to specific endotoxin as well as to the nonspecific mitogens Con A, PHA or PWM. There was also a marked alteration in lymphoid cell type and number in the spleen of the rats after continuous infusion of endotoxin, with a marked increase in plasma cell infiltration and germinal center formation. There was no significant alteration in glucocorticoid steroid levels. Control rats given saline only for a period of seven days via an implanted pump showed no or minimal effect for the first 5 days and then a delayed depression of blastogenic responses to LPS and to Con A, but this time lag contrasted markedly to the much earlier unresponsiveness of splenocytes from rats infused with endotoxin. Only a slight to moderate cellular infiltration occurred in the spleen of control rats implanted with a pump infusing saline only or an empty pump. Thus endotoxin infusion in a continuous manner via an implanted pump accounted for the early and marked suppression of responsiveness, as well as alteration in spleen size and cellularity.


Wound infection rates in clean surgery: a potentially misleading risk classification. To evaluate the incidence of wound infection in inguinal hernioplasties, incisional hernioplasties, splenectomies, and splenectomies performed in patients with hepatosplenic schistosomiasis, and to examine the relationship of surgical wound infection to antibiotic use, patient age, length of stay in the hospital prior to surgery, and the duration of the operation. Retrospective surveillance study. One thousand five hundred forty-two clean operations were analyzed. Comparing response (wound infection) and explanatory variables (age, length of hospital stay, duration of surgery, antibiotics, and surgery type), we found that age, use of antibiotics, and type of surgery were statistically significant, while length of hospital stay and duration of surgery were not significant. From these results, we can predict that the probability of wound infection in surgical patients considering these significant variables is lower for patients ages 14 to 30 years and higher for patients ages 31 to 60 years and lower for patients with prophylactic antibiotic use (up to 72 hours of use) and higher for patients with prolonged use (more than 72 hours); and lower for patients undergoing inguinal heria, followed in ascending order by nonschistosomotic patients undergoing splenectomy in schistosomotic patients.


"Through a drinking glass and what was found there": pseudocontamination of a hospital's drinking water. Examination of suspended macroscopic debris that was noted in hospital cafeteria drinking water over a prolonged period of time. A tertiary care hospital. A retrospective description of events. Conventional medical microbiology techniques were employed to determine if contamination of drinking water had occurred. Microscopic examinations of the debris showed live nematodes similar to Strongyloides stercoralis rhabditiform larvae and amoebic trophozoites. A culture of an ice container in the cafeteria yielded coliform bacteria. Hospital employees lost work because of a diarrheal illness thought to be contracted from drinking cafeteria water. Further investigation revealed that the debris contained numerous ciliated organisms, nematodes, fresh water amoebae, bacterial mats, flagellated fungi, and unidentified cysts suggesting that the debris was of a freshwater origin rather than fecal contamination. Another hospital served by a collateral city water line experienced a similar problem. The debris was observed in November when lake water in the reservoir undergoes inversion. Nonpathogenic freshwater microbiota probably originating from the city reservoir were the cause of pseudocontamination of hospital drinking water.


Role of protein kinase activation in the induction of B cell adhesion by MHC class II ligands. Engagement of MHC class II (Ia) molecules on B cells induces tyrosine phosphorylation, phosphoinositide turnover, elevation of intracellular calcium concentrations, and a rise in cAMP levels. However, a role for these biochemical signals in mediating functional responses induced by Ia ligands remains largely undefined. In this study, we utilized the induction of B cell adhesion by Ia ligands to demonstrate a role for signals transduced via Ia molecules in the generation of a functional response. Ia ligands that induced B cell aggregation induced tyrosine phosphorylation, whereas Ia ligands that did not induce B cell aggregation failed to induce any detectable tyrosine phosphorylation. Ia-induced B cell aggregation and tyrosine phosphorylation were inhibited by genistein and by herbimycin A, inhibitors of tyrosine kinases (PTK). Sphingosine and calphostin C, inhibitors of protein kinase C (PKC), also inhibited Ia-induced adhesion whereas HA1004, an inhibitor of cyclic nucleotide-dependent kinases, did not. Ia ligands induced both LFA-1-dependent and LFA-1-independent B cell adhesion. These two pathways of cell adhesion differed in their requirement for activation signals. PKC activation was sufficient for LFA-1-dependent adhesion, whereas LFA-1-independent adhesion required independent phosphorylation events mediated by PKC and by PTK. These results provide functional relevance for biochemical signals transduced via Ia molecules by demonstrating that Ia-induced B cell adhesion is mediated by the activation of PKC and by one or more PTK.


The beta 2-microglobulin dissociation rate is an accurate measure of the stability of MHC class I heterotrimers and depends on which peptide is bound. Stable, recombinant, water-soluble complexes of HLA-A2 and HLA-B27 were reconstituted from 125I-labeled beta 2-microglobulin (beta 2m), a synthetic peptide, and HLA H chain fragments expressed as inclusion bodies in the Escherichia coli cytoplasm. Using this system, we were able to show: 1) the t1/2 of beta 2m dissociation from HLA complexes at 37 degrees C varied from approximately 40 h to less than 1 h, depending on the peptide employed for reconstitution. Peptide length and composition were found to be critical factors in determining the beta 2m dissociation rate. Endogenous peptides form complexes that are about as stable as those formed with typical antigenic peptides. 2) Peptide exchange reactions, in which an exogenous peptide replaces the peptide that is already bound by the class I molecule, proceed readily for complexes that have rapid beta 2m dissociation rates. Thus, difficulties in demonstrating peptide binding to complexes that contain endogenous peptides can be attributed to the stability of the endogenous peptide/class I molecule complex. 3) The peptide exchange reaction does not require concomitant beta 2m dissociation. 4) Distal parts of the class I molecule, which are not directly involved in peptide binding or beta 2m binding, have a major impact on the stability of class I molecules. Thus, these studies show that the dissociation rate of beta 2m is an excellent measure of how tightly a given peptide binds to class I MHC molecules, that the ability to bind peptide is tightly coupled to the binding of beta 2m and vice versa, and that regions of the molecule distal from the binding site influence the stability of peptide binding.


Heterogeneity in antigen processing by different types of antigen-presenting cells. Effect of cell culture on antigen processing ability. The ability of normal B cells, peritoneal macrophages, and splenic APC to process and present OVA to a panel of T-T hybridomas with different specificities was investigated. In all cases, B cells were less efficient than unfractionated splenocytes in presenting OVA or its peptides. However, when the presentation of native Ag was compared to the presentation of peptides, it was obvious that there were marked differences in the ability of these two APC populations to generate different epitopes from OVA. Leupeptin inhibits the processing of selected epitopes from native OVA differently when it was presented by spleen cells or B cells, suggesting that these two APC populations differ in their protease content. The effect of in vitro culture on the ability of splenic and peritoneal APC to process OVA was also investigated. Native OVA presentation by macrophages and spleen cells was affected by in vitro culture, more for some epitopes than for other epitopes. In contrast, presentation of exogenous peptides by paraformaldehyde-fixed APC was either not affected by previous culturing for 3 days, or very much improved. Altogether, these data demonstrate that different epitopes on the same protein may be independently and differentially processed by B cells and spleen cells. Furthermore, the precise peptides that are produced may vary with the physiologic state of the APC.


A conditional secretory mutant in an Ig L chain is caused by replacement of tyrosine/phenylalanine 87 with histidine. The kappa-chain of the myeloma MOPC 21 is an unusual L chain, in that it is not secreted unless complexed with a H chain. This nonsecreted kappa-chain seems to be retained in the endoplasmic reticulum in association with the protein BiP/GRP78, both in myeloma cells and when expressed in COS-1 fibroblasts. By assaying the fate of the MOPC 21 kappa-chain and its mutated derivatives in COS-1 cells, we show that the cause of the nonsecreted phenotype is the presence of histidine in position 87 of the variable domain. When this amino acid is changed back to the tyrosine that usually occupies position 87, secretion of the unassembled kappa-chain is restored. As in B lymphoid cells, co-expression of gamma H chains in COS-1 cells complements the mutation in the L chain, and rescues secretion of the arrested kappa. Thus, the presence of histidine at position 87 creates a conditional L chain secretory mutant: it is not compatible with normal transport of free L chain, but can be rescued in the presence of H chain.


The HLA class I gene family includes at least six genes and twelve pseudogenes and gene fragments. We report the characterization of eight HLA class I homologous sequences isolated from cosmid and lambda libraries made from lymphoblastiod cell line 721 DNA. Four of these sequences, each contained within HindIII fragments of 1.7, 2.1, 3.0, and 8.0 kb, have class I homology extending over short intronexon regions. The remaining four are found within 7.5-, 8.0-, 9.0-, and 16.0-kb HindIII fragments, the first having homology to the 5' half of a class I gene whereas the latter three are homologous to the 3' portion of a class I gene. When combined with the characterization of other class I clones, this work brings the total number of HLA class I homologous sequences cloned and characterized to 18. Restriction mapping of cosmid clones showed that some of these sequences are linked to one another and to other class I pseudogenes and genes within 50-kb regions. Reconstruction experiments using the 18 class I genes and pseudogenes were performed that indicated that we had cloned all of the members of the HLA class I gene family detectable using HLA-A2 genomic DNA as probe. An additional 19th member of the class I gene family was identified using an HLA-E cDNA probe. Further Southern analysis with other class I probes indicated the 19 sequences comprise the entire class I gene family in LCL 721. Locus-specific probes were isolated from five of the eight clones and were used in Southern analysis of diverse genomic DNA to examine the polymorphism of the pseudogene sequences, demonstrating that some of them were highly polymorphic and some were missing entirely in certain haplotypes. An additional class I sequence, not contained within the 721 genome, was identified and may be found in association with the HLA-A11-Bw60 haplotype. Sequence comparisons were carried out to examine the evolutionary relationships among the pseudogenes. Hypothetical events in the evolution of the class I region are discussed.


Examination of four HLA class I pseudogenes. Common events in the evolution of HLA genes and pseudogenes. The HLA class I gene family in lymphoblastoid cell line 721 has been studied in detail and a number of sequences in addition to the classical genes have been identified. The cloning, characterization, and nucleotide sequences of four sequences, all full length HLA class I pseudogenes, are described in this report. These pseudogenes, contained within 5.4-, 5.9-, 7.0-, and 9.2-kb HindIII fragments, each have the class I exon-intron structure as well as class I homology in their 5' and 3' flanking regions. However, all four sequences have one or more substitutions that perturb the coding region, leaving little doubt that they are in fact pseudogenes. Comparisons among these sequences and the HLA class I genes revealed that their homology with the class I genes is patchwork. Thus, although some regions have diverged, other contiguous intron-exon sequences are highly conserved. Comparisons in the 5' regions indicate that the pseudogene promoters more closely resemble the classical HLA promoters than the nonclassical promoters as none of the unique structural features found in the HLA-E, -F, or -G regulatory regions are present in any of the pseudogene promoters. Further comparisons revealed that at least two putative gene conversion events, similar to those hypothesized to have occurred in the evolution of some HLA genes, may have occurred in the evolution of some of the pseudogenes. These and other hypothetical events in the evolution of the class I gene family are discussed.


Genomic structure and strain-specific expression of the natural killer cell receptor NKR-P1. NK cells are able to lyse a variety of virally infected and neoplastic cells in an MHC-unrestricted manner. The cell-surface protein NKR-P1 is thought to play a key role in this process. NKR-P1, initially identified in rat IL-2 activated NK cells, is encoded in the mouse by at least three similar, but not identical, genes. We previously reported the isolation and characterization of three different NKR-P1 cDNA, termed cDNA 2, 34, and 40, from IL-2 activated mouse NK cells. This report describes the structure of the gene encoding NKR-P1 cDNA 2, the smallest of these three cDNA. Gene 2 is composed of six exons spanning approximately 14 kb of genomic DNA. The first exon encodes the N-terminal intracellular domain, and exons 4, 5, and 6 contain the sequences coding for the CRD. This organization is similar to that of other genes that encode C-type animal lectins. The expression of the NKR-P1 genes in A-LAK cells from 13 mouse strains was examined by Northern blot analysis. NKR-P1 expression appears to coincide with that of the NK1.1 Ag. This observation further supports the hypothesis that the NK1.1 Ag is encoded by one of the NKR-P1 genes. Nucleotide sequence analysis of the promoter region of the three NKR-P1 genes in BALB/c and C57BL/6 mice suggests that differences in the level of expression probably do not result from alterations in the upstream regions of these genes, but may be caused by the expression of strain-specific transacting factors.


Locus-specific transcriptional control of HLA genes. One remarkable genetic feature of the class I MHC genes is their unparalleled degree of genetic polymorphism and diversity. The polymorphism is reflected by the fact that multiple loci encode class I molecules, and for each locus there are multiple alleles. In the course of investigating the regulation of HLA-A and HLA-B mRNA in human colorectal carcinoma cell lines, we have noticed a noncoordinate expression of the HLA mRNA in some of these cell lines. This observation prompted us to make use of these cell lines to study the locus-specific transcriptional regulation of HLA genes. Bandshift and footprint assays revealed at least three distinct and independent DNA-binding factors that bind to the core regulatory element of the HLA-A and HLB-B gene locus. A "novel" DNA-binding factor recognizing the CCAAT motif seems to be important for locus-specific expression of HLA-A mRNA, whereas a different factor which binds to a Sp1-like sequence is crucial for normal HLA-B mRNA expression. In certain colorectal cancer cell lines, underrepresentation of these locus-specific DNA-binding proteins correlates with the locus-specific down-regulation of HLA mRNA. This observation is further supported by experiments which demonstrated that the locus-specific suppression of exogenously introduced TK-chloramphenicol acetyltransferase DNA constructs, containing the "putative" HLA locus-specific DNA core regulatory sequence, is regulated in a locus-specific manner when introduced into these HLA-A- and HLA-B-deficient human colorectal cell lines.


IL-7 up-regulates the expression of IL-8 from resting and stimulated human blood monocytes. Blood monocytes are important cellular sources of a vast array of bioactive substances, including regulatory and chemotactic cytokines. The regulation of these cytokines is of critical importance to the expression of acute and chronic inflammatory responses. IL-7, a T and B cell-activating cytokine, has recently been shown to have stimulatory effects on the expression of several monocyte-derived proinflammatory cytokines. We now describe the induction of IL-8 mRNA and extracellular protein from human blood monocytes by IL-7. The up-regulation of IL-8 mRNA by IL-7 was not altered by concomitant treatment with cycloheximide, suggesting that the direct stimulatory effects of IL-7 were not dependent upon de novo protein synthesis. In addition, IL-7 significantly potentiated the production of IL-8 from LPS-, TNF-, and IL-1-treated peripheral blood monocytes. Our findings suggest that IL-7 may play a critical role in the modulation of macrophage-derived cytokine expression and may function in vivo as an important proinflammatory cytokine.


IFN-gamma and 1,25(OH)2D3 induce on THP-1 cells distinct patterns of cell surface antigen expression, cytokine production, and responsiveness to contact with activated T cells. Differentiation and maturation of monocytes are accompanied by the expression of specific surface glycoproteins, the secretion of cytokines, and the capacity to respond to ligands. These changes may be influenced by interactions with hormones, soluble lymphocytic products, or direct contact with lymphocytes. We have studied two distinct pathways in the differentiation of a human monocytic cell line, THP-1: one being induced by IFN-gamma and the other by 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). In THP-1 cells, IFN-gamma induces cell surface expression of HLA-DR and CD54 and production of IL-1 beta, TNF-alpha, and IL-6. In contrast, 1,25(OH)2D3 increases cell surface expression of CD11b and CD14, but fails to stimulate cytokine production. Direct contact of THP-1 with stimulated fixed T cells markedly induces IL-1 beta, TNF-alpha, and IL-6 production by THP-1. Production is higher when THP-1 have been previously exposed to 1,25(OH)2D3 as compared to prior exposure to IFN-gamma. mAb raised against certain relevant cell surface glycoproteins on THP-1 were tested for their ability to block the response of THP-1 to T cells. Antibodies to CD11a, CD11b, and CD11c, alone or in combination, only partially blocked IL-1 beta production by THP-1, whereas antibodies to CD54 and CD14 did not. Thus other unknown structures on the THP-1 cells may be involved in the induction of THP-1 cytokine production by T cell contact.


Adherence of infected erythrocytes to venular endothelium selects for antigenic variants of Plasmodium falciparum. Erythrocytes (E) infected with asexual forms of malaria parasites exhibit surface antigenic variation. In Plasmodium falciparum infections, the variant Ag is the P. falciparum E membrane protein 1 (PfEMP1). This molecule may also mediate the adherence of infected E to host venular endothelium. We show here that parasite lines selected for increased adherence to endothelial cells have undergone antigenic variation. Three adherent lines selected from the same P. falciparum clone reacted with the same agglutinating antiserum that failed to agglutinate the parental clone. Immunoprecipitation experiments with the agglutinating anti-serum demonstrated that the selected lines expressed cross-reactive forms of PfEMP1 that were of higher m.w. and antigenically distinct from PfEMP1 of the parental clone. When one of the adherent lines was cloned in the absence of selection, a range of variant antigenic types emerged with differing cytoadherence phenotypes. These findings show that selection for cytoadherence in vitro favors the emergence of antigenic variants of P. falciparum and suggest that the requirement for cytoadherence in vivo may restrict the range of antigenic variants of P. falciparum in natural infections.


Nongranular proteolytic enzymes of rat IL-2-activated natural killer cells. I. Subcellular localization and functional role. Our investigations indicate that a variety of neutral serine proteases exist in highly purified, IL-2-activated rat NK (A-NK) cells. These enzymatic activities are not restricted to only cytolysin-containing granules and are not defined by only the assay of N-alpha-benzyloxycarbonyl-L-lysine thiobenzylesterase activity. These activities, which we term A-NKP 1, A-NKP 2, A-NKP 3, and A-NKP 4, cleave, respectively, the following fluorogenic peptide substrates: Boc-Phe-Ser-Arg-7-amino-4-methylcoumarin (AMC, trypsin-like); Suc-Ala-Ala-Phe AMC (chymotrypsin-like); Suc-Gly-Pro-Leu-Gly-Pro AMC (collagenase-like), and Z-Phe-Arg AMC (another trypsin-like enzyme). The proteases A-NKP 1, A-NKP 2, and A-NKP 3 are not cell surface-associated and appear to be cytosolic as defined by isopycnic sucrose density gradient centrifugation. In contrast, A-NKP 4 appears to be located in lysosomes. Treatment of rat A-NK cells with protease inhibitors that inhibit A-NKP 2 and A-NKP 3 also substantially inhibit A-NK cell-mediated cytotoxicity against both NK-sensitive and -resistant targets (YAC-1 and P815, respectively). These results indicate that A-NKP2 and A-NKP 3 may play a role in IL-2-activated NK cell-mediated cytotoxicity. A variety of proteolytic enzymes, in addition to granzymes, therefore exist in A-NK cells. Our studies indicate that a prerequisite to a thorough understanding of the role of proteases in killer cell function is the investigation of several classes of enzymes in addition to granzymes contained in lytic granules.


In vivo activity of tumor necrosis factor (TNF) mutants. Secretory but not membrane-bound TNF mediates the regression of retrovirally transduced murine tumor. We have previously demonstrated that murine tumor cells transduced with a retrovirus containing the cDNA encoding wild-type human TNF regress in vivo when injected into immunocompetent mice; this regression is T cell mediated. To determine whether membrane-associated or secreted TNF was responsible for tumor regression, we transduced a cloned murine fibrosarcoma 205 F4 with retroviruses encoding modified human TNF genes. The cloned tumor lines of one retroviral transduction expressed only membrane bound 26-kDa TNF. This TNF could not be cleaved or secreted, but was present on the cell surface. A second retrovirus caused the expression of only secretory 17-kDa TNF, as the transmembrane domain of the cDNA was deleted. The TNF produced by tumor cells transduced with either retroviral vector was functional in vitro as direct lysis of the TNF-sensitive target L929 by transduced tumor cells was demonstrated. The TNF present on 26-kDa expressing tumors was membrane bound as supernatants from cultured 17-kDa TNF expressing tumor cells but not 26-kDa TNF expressing tumors mediated the lysis of L929 cells. Both tumors were injected s.c. into syngeneic mice and tumor growth was measured serially. In repeated experiments, 26-kDa TNF expressing tumors grew progressively in all mice. In contrast, 17-kDa TNF expressing tumors grew for 10 days and then regressed with all animals free of tumor at 28 days. Tumor regression was abrogated by in vivo injection of an anti-TNF antibody. Similar results were obtained in a second tumor model, 203 E4. Thus regression of TNF transduced tumors in vivo requires secretion of TNF, as membrane-bound TNF is insufficient to elicit the host response.


Diphtheria toxin- and Pseudomonas A toxin-mediated apoptosis. ADP ribosylation of elongation factor-2 is required for DNA fragmentation and cell lysis and synergy with tumor necrosis factor-alpha. We have reported that diphtheria toxin (DTX) mediates target cell lysis and intranucleosomal DNA fragmentation (apoptosis) and also synergizes with TNF-alpha. In this paper, we examined which step in the pathway of DTX-mediated inhibition of protein synthesis was important for induction of cytolytic activity and for synergy. Using a DTX-sensitive tumor cell line, we first examined the activity of the mutant CRM 197, which does not catalyze the ADP ribosylation of elongation factor-2 (EF-2). CRM 197 was not cytolytic for target cells and did not mediate intranucleosomal DNA fragmentation of viable cells. The failure of CRM 197 to mediate target cell lysis suggested that the catalytic activity of DTX is prerequisite for target cell lysis. This was corroborated by demonstrating that MeSAdo, which blocks the biosynthesis of diphthamide, inhibited DTX-mediated protein synthesis inhibition and also blocked target cell lysis. Furthermore, the addition of nicotinamide, which competes with NAD+ on the DTX action site of EF-2, also blocked DTX-mediated lysis. These findings suggest that ADP-ribosylation of EF-2 may be a necessary step in the pathway leading to target cell lysis. In contrast to the sensitive line, the SKOV-3 tumor cell line is sensitive to protein synthesis inhibition by DTX but is not susceptible to cytolysis and apoptosis by DTX. Thus, protein synthesis inhibition by DTX is not sufficient to mediate target cell lysis. The synergy in cytotoxicity obtained with the combination of DTX and TNF-alpha was examined in order to determine the pathway mediated by DTX in synergy. Like the direct lysis by DTX, synergy was significantly reduced by MeSAdo and by nicotinamide. Furthermore, synergy was not observed with combination of CRM 197 and TNF-alpha. These results demonstrate that, in synergy, DTX may utilize the same pathway required for its cytolytic activity. Pseudomonas aeruginosa exotoxin shared most the properties shown for DTX. Altogether, these findings demonstrate that DTX-mediated apoptosis is initiated at a step beyond the ADP ribosylation of EF-2.


Selective suppressive effects of Trypanosoma cruzi infection on IL-2, c-myc, and c-fos gene expression. Infection with Trypanosoma cruzi is accompanied by a profound suppression of immune responses including the production of IL-2. Previous experiments have confirmed a correlated decrease in IL-2 mRNA levels in lymphoid cells from infected mice. To further define the molecular basis of this regulation, we have examined the production and degradation of mRNA for IL-2 and other T cell activation genes in cells from T. cruzi-infected mice. Spleen cells from C57BL/6J mice infected with the Brazil strain of T. cruzi were analyzed for the kinetic expression of IL-2, IL-2R alpha, c-myc, and c-fos genes in response to Con A and PMA costimulation. Cells from infected mice exhibited a selective reduction of c-myc and c-fos mRNA in association with the severe suppression of the IL-2 gene, but a less severe to comparable production of IL-2R alpha mRNA compared with normal spleen cells. The similar patterns of the suppression of c-myc and IL-2 mRNA suggest a common mechanism of down-regulation of these two genes in T. cruzi infection. Actinomycin D treatment was used to demonstrate that decreased steady state levels of IL-2, c-myc, and c-fos mRNA in cells from infected mice were not due to an increased rate of degradation of these mRNA. Cycloheximide treatment enhanced the expression of IL-2, IL-2R alpha, c-myc, and c-fos mRNA in spleen cells from both normal and infected mice. Although a larger percentage of induction was observed in cells from infected mice, the mRNA levels for IL-2, c-myc, and c-fos in cells from infected mice were still lower than those of normal cells. Spleen cells from infected mice precultured for 24 to 72 h before the addition of mitogens showed significant enhancement of IL-2 and c-myc gene expression; however, this recovery was inhibited if fixed T. cruzi was present in the preculture medium. These data suggest that the reduction of IL-2 mRNA in infected mice is not the result of an increased degradation of its mRNA but to down-regulation of transcription of the IL-2 gene in T cells from T. cruzi-infected mice. Preculture-induced recovery of IL-2 production appears to result from release from this regulation and full expression of the IL-2 gene.


A T cell clone directed at the circumsporozoite protein which protects mice against both Plasmodium yoelii and Plasmodium berghei. Clone B is a cytotoxic T cell clone induced by immunization with Plasmodium yoelii sporozoites which recognizes an epitope on both the P. yoelii and Plasmodium berghei circumsporozoite proteins. It is CD8, uses the V beta 8.1 TCR, and is Kd restricted. When adoptively transferred, it protects mice against infection by both species of malaria sporozoites, and this protection is dependent on IFN-gamma. Clone B cells are more broadly reactive and protective than previously described murine T cell clones against malaria. Clone B may be an important model for immune protection against the spectrum of variant parasites in nature.


IL-10 induces transcription of the gene for mouse mast cell protease-1, a serine protease preferentially expressed in mucosal mast cells of Trichinella spiralis-infected mice. Mouse bone marrow-derived mast cells (BMMC) obtained by culturing progenitor cells with rIL-3 express mouse mast cell protease (MMCP)-5 mRNA but not MMCP-1 mRNA or MMCP-4 mRNA. In terms of mast cell differentiation, these transcripts encode one early-expressed and two late-expressed chymases, respectively. cDNA and cRNA probes were used in RNase protection assays and RNA blot analyses to study the expression of these three homologous protease genes in cultured mast cells and in helminth-infected mice. Intestinal tissue from Trichinella spiralis-infected mice, containing high numbers of mucosal mast cells, had abundant amounts of MMCP-1 mRNA but only minimal amounts of the serosal mast cell transcript that encodes MMCP-4. Exposure of mouse BMMC to rIL-10-induced transcription of the MMCP-1 gene but not the MMCP-4 gene, and a cDNA encoding MMCP-1 was obtained from these rIL-10-treated cells. The expression of MMCP-1 mRNA in BMMC depended on the continuous exposure of these cells to rIL-10, and the level of MMCP-1 mRNA (but not MMCP-5 mRNA) was substantially higher in BMMC maintained in rIL-4 and rIL-10 than in rIL-3 and rIL-10 or in rIL-3, rIL-4, and rIL-10. Thus, whereas rIL-3 elicits transcription of early expressed genes in cultured mast cells, it suppresses the transcription of late-expressed genes. These in vitro and in vivo transcription studies also indicate that rIL-10 preferentially induces differentiation of mouse progenitor cells in a mucosal mast cell-specific lineage, and that expression of granule serine protease genes is regulated in a subclass-specific manner in mouse mucosal mast cells and serosal mast cells.


Tumor target-derived soluble factor synergizes with IFN-gamma and IL-2 to activate macrophages for tumor necrosis factor and nitric oxide production to mediate cytotoxicity of the same target. Inflammatory mouse peritoneal macrophages were activated by IFN-gamma in synergy with IL-2 or Lipid A to mediate TNF production for autocrine generation of cytotoxic nitric oxide (NO) to kill P815 or L1210 tumor targets. It was determined that for IL-2, but not Lipid A, to effectively trigger activation of IFN-gamma-primed macrophages, the tumor targets must be also present for interaction with effector macrophages to mediate the production of TNF and NO. IFN-gamma- and IL-2-activated macrophages from syngeneic DBA/2 and allogeneic C3H mice had identical MHC-unrestricted requirements for interaction with DBA/2 mouse-derived P815 and L1210 targets to mediate production of TNF and NO for tumor cytotoxicity. To further define the mechanistic requirements for macrophage-tumor target interaction, IFN-gamma- and IL-2-activated macrophages were separated from P815 targets in culture by a semipermeable membrane. Under these conditions, both TNF and NO were produced by the macrophage, which indicated that the requirement for tumor target-macrophage interaction may be due to a soluble factor produced by the target rather than to direct physical contact. This was confirmed by experiments in which 24-h cell-free culture fluids, derived from either P815 or L1210 tumor targets, substituted for the intact tumor cells in the stimulation of TNF mRNA synthesis and secretion with NO generation of TNF mRNA synthesis and secretion with NO generation by IFN-gamma- and IL-2-activated C3H or DBA/2 macrophages. The activity in 24-h culture fluids derived from P815 and L1210 tumor targets was tentatively designated as tumor-derived recognition factor(s) (TDRF) since it was produced constitutively by the tumor targets and synergized with IFN-gamma and IL-2 to induce macrophage production of TNF and NO for death of the same targets. A variety of nontransformed human and mouse fibroblasts, mouse spleen lymphocytes, and two adherent mouse fibrosarcomas did not produce detectable TDRF activity, whereas two mouse T lymphomas, EL4 and EL4.IL-2, produced TDRF activity similar to L1210 mouse leukemia and P815 mastocytoma. The C3H/MCA, a TDRF-nonproducing mouse fibrosarcoma, was susceptible to cytotoxicity mediated by macrophages activated by IFN-gamma and Lipid A, but not by IL-2 triggering. Exogenous TDRF derived from L1210 targets reconstituted the cytotoxic activity for C3H/MCA MCA targets mediated by IFN-gamma- and IL-2-activated macrophages accompanied by the production of TNF and cytotoxic NO.(ABSTRACT TRUNCATED AT 400 WORDS)


A threshold level of coupled G-proteins is required to transduce neutrophil responses. Chemoattractant-induced activation of human polymorphonuclear leukocytes involves receptor coupling to guanine nucleotide binding proteins (G-proteins). Treatment of polymorphonuclear leukocytes with pertussis toxin, which ADP-ribosylates neutrophil G-proteins and uncouples G-proteins from receptors, causes a conversion of cells from responders to nonresponders rather than a gradual decrease in the ability of all cells to respond (Omann, G. M., and J. M. Harter. 1991. Cytometry 12:252; Omann, G. M., and M. M. Porasik-Lowes. 1991. J. Immunol. 146:1303). Flow-cytometric methods were used to measure N-formylpeptide-induced cytosolic Ca2+ elevation and actin polymerization over a wide range of ADP-ribosylation levels and showed that although the percentage of responding cells varied markedly, the responding cells were stimulated equivalent to controls. The conditions of pertussis toxin (PT) treatment did not interfere with non-G-protein-mediated pathways as assessed by measurement of phagocytosis, a complex process involving the cytoskeleton. We tested the explanation that the all-or-none effect may have been due to heterogeneous insertion of the catalytic subunit of PT into the cells such that responders had no ADP-ribosylation and nonresponders were completely ADP-ribosylated. Measurement of the binding of fluorescent N-formylpeptides to permeabilized cells, which allows the distinction between completely ribosylated and normal cells, showed that all cells treated with a submaximal concentration of PT had intermediate levels of receptor-coupled G-proteins. Thus, partial ADP-ribosylation had occurred in all cells and the all-or-none insertion of the catalytic subunit of PT was ruled out. Thus, there is a threshold of coupled G-proteins required to transduce responses. The ability of PT to inhibit N-formylpeptide-induced actin polymerization and cytosolic calcium elevation was compared and showed that both responses have essentially the same threshold of G-proteins required to transduce the responses. Thus, the pathways regulating actin polymerization and calcium elevation appear to be coupled with equal efficiency to the G-proteins.


Production of nitric oxide by murine bone marrow cells. Inverse correlation with cellular proliferation. The present studies were designed to assess the ability of primary cultures of bone marrow cells to produce nitric oxide. We found that two inflammatory stimuli, IFN-gamma and LPS, were potent inducers of nitric oxide production by bone marrow cells. In addition, the CSF granulocyte-macrophage (GM)-CSF and IL-3 as well as TNF-alpha, while inactive by themselves, were synergistic with LPS and IFN-gamma in inducing nitric oxide production. Maximal effects were observed with combinations of GM-CSF and LPS. Nitric oxide production by bone marrow cells was found to be dependent on the presence of L-arginine in the culture medium and inhibitable by NG-monomethyl-L-arginine and L-canavanine, two nitric oxide synthase inhibitors. Nitric oxide produced by the cells was also suppressed by TGF-beta 1 and the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. Separation of bone marrow cells by density gradient centrifugation and flow cytometry revealed that the granulocyte-containing fraction was largely responsible for nitric oxide production. In additional experiments we found that treatment of bone marrow cells with GM-CSF significantly stimulated bone marrow cell growth. In contrast, the combination of GM-CSF and LPS or IFN-gamma markedly suppressed cellular proliferation. This suppression was completely reversed by treatment of the cells with NG-monomethyl-L-arginine. Taken together, these data demonstrate that various inflammatory stimuli and cytokines induce nitric oxide production by primary cultures of bone marrow cells and that this mediator may play a role in the regulation of bone marrow cell growth and development.


Induction of granulomatous experimental autoimmune thyroiditis in mice with in vitro activated effector T cells and anti-IFN-gamma antibody. Experimental autoimmune thyroiditis (EAT) can be induced in mice after the transfer of mouse thyroglobulin (MTg)-sensitized donor spleen cells that have been activated in vitro with MTg. CD4+ T cells are required for the transfer of EAT in this model. Because CD4+ T cells produce various lymphokines, such as IFN-gamma, that may be involved in the activation or regulation of the immune response to MTg and the development of EAT, the present study was undertaken to determine whether a neutralizing mAb to IFN-gamma could modulate the induction or expression of EAT. The anti-IFN-gamma mAb XMG-1.2 had no effect on sensitization of donor cells. However, addition of XMG-1.2 mAb during in vitro activation of MTg-primed spleen cells resulted in more severe EAT in recipient mice. The thyroid lesions in recipients of cells cultured with MTg and XMG-1.2 mAb also exhibited granulomatous changes, which differed qualitatively from the predominantly lymphocytic cell infiltrates in recipients of cells cultured with MTg alone. Recipients of MTg-activated spleen cells also developed severe granulomatous EAT when they were given injections of XMG-1.2 mAb. The effects of XMG-1.2 could be neutralized by IFN-gamma. Recipients of cells cultured in the presence of XMG-1.2 mAb had augmented autoantibody responses, although there were no apparent differences in the IgG subclass distribution of the anti-MTg autoantibody responses. These studies suggest that neutralization of endogenous IFN-gamma results in increased activity of cells capable of inducing granulomatous EAT in mice.


A human systemic lupus erythematosus-related anti-cardiolipin/single-stranded DNA autoantibody is encoded by a somatically mutated variant of the developmentally restricted 51P1 VH gene. We report the Ig H and L chain V region sequences from the cDNAs encoding a monoclonal human IgG anti-cardiolipin/ssDNA autoantibody (R149) derived from a patient with active SLE. Comparison with the germ-line V-gene repertoire of this patient revealed that R149 likely arose as a consequence of an Ag-driven selection process. The Ag-binding portions of the V regions were characterized by a high number of arginine residues, a property that has been associated with anti-dsDNA autoantibodies from lupus-prone mice and patients with SLE. The VH gene encoding autoantibody R149 was a somatically mutated variant of the 51P1 gene segment, which is frequently associated with the restricted fetal B cell repertoire, malignant CD5 B cells, and natural autoantibodies. These data suggest that in SLE patients a common antigenic stimulus may evoke anti-DNA and anti-cardiolipin autoantibodies and provide further evidence that a small set of developmentally restricted VH genes can give rise to disease-associated autoantibodies through Ag-selected somatic mutations.


Amplified ELISPOT assay for the detection of HIV-specific antibody-secreting cells in subhuman primates. A novel immunoenzyme amplification technique has been evaluated in an ELISPOT assay for the detection of antigen-specific antibody-secreting cells (ASC) in monkeys. In this assay, mononuclear cells containing putative ASC are incubated for a few hours in antigen-coated wells. Following removal of the cells, zones of solid phase bound antibodies secreted by individual ASC are visualized in four consecutive steps. First, a primary biotinylated anti-immunoglobulin (Ig) reagent is added followed by enzyme-labelled avidin. The amplification procedure comprises the addition of biotinylated anti-enzyme antibodies in the third stage, followed by enzyme-conjugated avidin and substrate. When evaluated in a modified ELISPOT assay for the detection of simian B cells secreting antibodies to the envelope glycoprotein gp120 of the human immunodeficiency virus type 1 (HIV-1), this amplification procedure proved to be suitable even when using anti-human Ig antisera as primary antibody reagents. This development should be useful for other ELISPOT assays where species specific anti-Ig reagents are not always available and, most importantly, for enumerating cells producing immunoreactive substances in such minute amounts that they may escape detection by conventional ELISPOT assays. Furthermore, a functional simian HIV-specific ELISPOT assay could prove valuable for assessing the humoral immunogenicity of future candidate vaccines against the acquired immunodeficiency syndrome (AIDS).


Influence of collection and separation of blood samples on plasma IL-1, IL-6 and TNF-alpha concentrations. The anticoagulant used for the collection of blood was found to influence in vitro cytokine production in whole blood. Lithium heparin in certain collection tubes was found to contain endotoxin and induced cytokine synthesis in a time-dependent manner whereas endotoxin-free lithium heparin did not. No induction of cytokine occurred in the presence of EDTA which was also able to inhibit endotoxin-induced cytokine synthesis. Synthesis or absence of cytokine correlated with the induction of messenger RNA. Investigation of the kinetics of cytokine induction in whole blood revealed that tumour necrosis factor alpha (TNF) was detectable after 2 h of incubation at 37 degrees C and interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) after 3 h. In certain samples IL-1 and IL-6 were detectable in plasma separated immediately from blood collected into endotoxin-free lithium heparin, presumably reflecting in vivo synthesis, and similar concentrations were detected after 3 h of incubation of whole blood at 37 degrees C. These data indicate that as long as blood is collected into endotoxin-free anticoagulant then cytokine measurements will reflect the in vivo status.


A simple and economical strategy for downstream processing of specific antibodies to human transferrin from egg yolk. A rapid and economical procedure for extraction of antibodies from egg yolk is described. Laying hens were immunized with human transferrin and extracts of egg-yolk were purified with a procedure based on affinity chromatography. The resulting purified antibodies were evaluated in a nephelometric system for the assay of transferrin in human sera. The results agreed closely with those obtained with a commercially available anti-human transferrin serum from rabbits.


Cellular enzyme-linked immunocircle assay. A rapid assay of hybridomas produced against cell surface antigens. A novel method has been developed for the initial screening of hybridomas produced against cell surface antigens. Glutaraldehyde-fixed cells were immobilized as targets on the lid of a 96-well tissue culture plate which had been precoated with poly-L-lysine. Antibody binding was determined by an immunoenzymatic method in an arrangement permitting both macro- and microscopic examination. After optimization with mouse thymus cells using existing rat monoclonal antibodies, new rat-mouse hybridoma cell lines against mouse thymocytes and bone marrow cells were screened. The antibodies could be characterized immediately both by the localization of the immune reaction (surface or intracellular) or as estimated by the frequency of positive cells recognized by the antibody in the sample.


The measurement of transforming growth factor type beta (TGF beta) levels produced by peripheral blood mononuclear cells requires the efficient elimination of contaminating platelets. In recent years there has been an increasing interest in measuring the levels of TGF beta produced by peripheral blood mononuclear cells (PBMC), since its abnormal regulation seems to be involved in several pathological states. Platelet-contamination, a common feature in PBMC populations isolated by the standard Ficoll-Paque method, would theoretically disturb the measurement of the levels of TGF beta produced by mononuclear cells, since platelets represent an important source of this cytokine. In this study, supernatants of PBMC cultures from healthy subjects, either platelet-contaminated or uncontaminated, were assayed for TGF beta activity in three different bioassays. We report that the presence of platelets led in most cases to an important overestimation of the TGF beta levels produced by MNC in the Swiss-3T3 bioassay and in a PBMC proliferation assay. In contrast, in the Mv1Lu bioassay these levels were significantly underestimated, an effect which we attribute to the presence of other platelet-derived growth factors. These results suggest that the elimination of platelets from PBMC cultures is essential if TGF beta production by mononuclear cells is to be studied.


Comparison of selected protocols for the generation of IgA isotype monoclonal antibodies against the gut parasite, Eimeria tenella. Investigations into the role of mucosal IgA in the protective immune response to alimentary tract parasites could be facilitated by the production of specific IgA monoclonal antibodies. To this end, we sought to generate IgA hybridomas against sporozoites of the protozoan, Eimeria tenella using different protocols. Of the methods investigated, intra-enteric priming followed by intravenous boosting of germ-free mice, using splenic lymphocytes in fusions, optimised the yield of IgA hybridomas. Using this protocol, 7/27 specific anti-Eimeria hybridoma antibodies isolated were of IgA isotype. When lymphocytes from mesenteric lymph nodes (MLN) were used in fusions more non-specific IgA secretors were produced than when splenic lymphocytes were used, but the yield of specific anti-Eimeria IgA secreting hybridomas was not improved. By all protocols, a total of nine IgA secreting hybridomas were identified of which eight have been cloned for further studies.


Specificity of three anti-complement factor 3 monoclonal antibodies. Monoclonal antibodies which recognize specific C3 fragments may be used to distinguish C3 cleavage products bound to organisms. We defined the specificity of three commercially available monoclonal antibodies by Western immunoblot analysis, enzyme-linked immunosorbent assay, and a quantitative flow cytometric technique. Two monoclonal antibodies with specificity for (i) an erythrocyte-bound C3d epitope or (ii) an erythrocyte-bound C3c epitope retained their specificity in all assays. However, the third monoclonal antibody with selectivity for erythrocyte-bound C3bi failed to retain specificity for C3bi bound to non-erythrocyte surfaces in each of our assays; binding instead to all C3 fragments containing the C3g domain. We postulate that erythrocyte C3b-binding surface proteins may alter the availability of certain C3b epitopes and influence observed anti-C3 monoclonal antibody specificity. We conclude that the specificity of monoclonal antibodies for C3 fragments should be confirmed with assays which do not employ erythrocytes or other surfaces bearing C3 receptors. Also, our quantitative flow cytometric technique is a potentially valuable tool for the enumeration of particle-bound C3 fragments.


Detection of fecal blood by colloidal gold agglutination using an anti-human hemoglobin monoclonal antibody. Monoclonal antibodies to human hemoglobin were produced and a colloidal gold agglutination method has been developed for detection of fecal occult blood. Since hemoglobin is composed of the tetramer, alpha 2 beta 2, a single monoclonal antibody-labeled colloidal gold can agglutinate with hemoglobin. The lowest detectable hemoglobin concentration was 0.5 micrograms/ml. A total of 785 fecal samples were determined using colloidal gold agglutination and compared with latex agglutination. The colloidal gold agglutination detected blood in 75 samples, whereas latex agglutination detected blood in 76 samples, and among them 70 were positive in both methods. Overall agreement between the two methods was 98%.


In vivo primary induction of virus-specific CTL by immunization with 9-mer synthetic peptides. A primary cytotoxic T lymphocyte (CTL) response in vivo requires antigen presentation by cytosolic processing and can not in general be obtained by vaccination with soluble proteins. In the present work we have found that vaccination of mice with pre-processed synthetic peptides, corresponding to endogenous 9-mers produced in influenza A virus-infected cells, resulted in strong primary CTL responses. The generated CTL efficiently killed virus-infected target cells with preference for viral strains having the identical amino acid sequences to the peptides used for immunization. The optimal conditions for a primary in vivo CTL response was obtained with 100 micrograms peptide dissolved in incomplete Freund's adjuvant and injected s.c. at the base of tail. Spleen cells which had been primed 7-10 days earlier were restimulated for 5 days in vitro, using an optimal low peptide concentration (0.05 microM) and tested against virus-infected and peptide-treated target cells. The peptide-induced CTL were major histocompatibility complex class I restricted and CD8 positive.


Monocyte proliferation in a cytokine-free, serum-free system. The aim of this study was to establish a cytokine-free, serum-free system which would enable the long-term survival and proliferation of human peripheral blood monocytes. Monocytes were isolated from peripheral blood mononuclear cells (PBMC) by adherence to untreated plastic petri dishes and maintained up to 6 weeks in serum-free medium (SFM) consisting of IMEM, insulin, transferrin, sodium selenite and BSA. Maximal cell proliferation occurred during the first 2 weeks of culture and corresponded to the appearance of large numbers of pure, nonadherent culture-derived macrophages. Monocyte maturation was characterised by the modulation of specific cell surface antigens. The percentage of cells staining for the transferrin receptor increased with time, whereas the percentages of cells expressing CD11b, CD11c and HLA-DR remained greater than 60% for the 15 days studied. The mean fluorescent intensities (MFI) of all these antibodies increased significantly with time. The only differences found between the adherent and nonadherent cells, using the above antibodies, were with the MFI for CD11b and CD11c. In both cases, the intensity of staining was significantly greater in the adherent cells. Estimation of cytokine production by cells maintained for 5 weeks in SFM found that they constitutively produced large amounts of macrophage colony-stimulating factor (M-CSF) in the absence of any exogenous stimuli. These cells were also found to secrete high levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) during the 1st week and granulocyte macrophage colony-stimulating factor (GM-CSF) during the 3rd week. However, the addition of exogenous GM-CSF (5 U/ml, S5) was found to significantly inhibit monocyte proliferation up to 17 days. This is the first report of proliferation associated with long-term survival of culture derived macrophages in a serum-free, cytokine-free system.


New parental cell lines for generating human hybridomas. In contrast to the success achieved with the production of hybridomas in the mouse system, creating human hybridomas is problematic. The reason is believed to be a lack of suitable malignant human cell lines. The work presented here demonstrates the establishment of three human parent cell lines--two of which are of T cell origin--by installing hypoxanthine guanosine phosphoribosyl transferase (HGPRT) and/or thimidine kinase (TK) deficiencies into the leukemic cell lines REH, 1301 and SKW-3. In order to isolate true hybridomas, selection procedures must guarantee complete death of the enzyme-deficient malignant parent cells. In this respect sublines with a combined HGPRT and TK deficiency proved to be superior to those with only one enzyme deficiency, especially in combination with the newly developed hypoxanthine/aminopterine/thymidine/azaserine (HATA) selection medium. However, selection media should be of low nonspecific toxicity. This was shown to be a particular property of the thymidine-free azaserine/hypoxanthine (AH) selection medium. Preliminary data show an extraordinary ability of one subclone of the hypoxanthine guanosine phosphoribosyl transferase-negative T cell line SKW-3 to generate human T-T hybridomas. They are of a stable, nearly tetraploid karyotype and express new surface antigens, thus providing new possibilities for the investigation of human T lymphocyte function by means of hybridoma technology.


An ultrasensitive system to detect IL-4: enzyme-linked immunosorbent assay (ELISA) combined with an avidin-biotin and enzyme amplification system. We established an ultrasensitive interleukin-4 enzyme-linked immunosorbent assay by combining ELISA with an avidin-biotin and enzyme amplification system. The resultant system (AB-EA ELISA) was 250 times more sensitive than conventional ELISA and 2.5 times more sensitive than enhanced ELISA using an enzyme amplification system alone. The ultrasensitive assay was specific to IL-4 alone; there was no cross reaction with other cytokines. Using the ultrasensitive assay, we measured IL-4 synthesis in vitro by unstimulated and stimulated peripheral blood mononuclear cells (PBMC) from patients with allergic rhinitis. PBMC from patients spontaneously produced measurable amounts of IL-4, whereas IL-4 production from PBMC of normal controls, if any, was below detectable levels. Stimulation of the cultures with LPS significantly increased IL-4 production in two of six patient PBMC cultures but in none of the control cultures; stimulation with Con A markedly increased IL-4 production in all patient PBMC cultures but in only two of seven control cultures. These results suggest that the AB-EA ELISA is a useful method to study the mechanism of IL-4 synthesis in type-I allergic diseases.


13C-NMR spectral analysis of the structures of mouse immunoglobulin G1 carrying allotypes a and j. A novel 13C nuclear magnetic resonance (NMR) method is described for the detection of subtle structural differences between mouse immunoglobulins carrying different allotypes. Fc fragments of mouse IgG1 antibodies carrying allotypes a and j have been selectively labeled with [1-13C]methionine. 13C-NMR spectra have shown that the microenvironment around Met-398 is significantly different for the two kinds of allotypes. Peptide mapping and amino acid sequence analyses have revealed that Val-406 of IgG1 carrying allotype a is substituted for Ile in the case of allotype j. X-ray crystallographic data indicate that Met-398 is in close spatial proximity to Val (Ile)-406. We therefore conclude that the 13C-NMR method can provide us with a novel spectroscopic probe for the structural characterization of allotypic markers.


Effective method for synthetic peptide immobilization that increases the sensitivity and specificity of ELISA procedures. A procedure is described for the immobilization of synthetic peptide antigens on a plastic solid phase for performing ELISA. The use of a streptavidin-biotinylated peptide system for coating microplates with peptide antigen markedly increased both the sensitivity and the specificity compared to a standard ELISA based on synthetic peptides. The procedure was used for the detection of HIV-1-specific antibodies.


A statistical model and computer program to estimate association constants for the binding of fluorescent-labelled monoclonal antibodies to cell surface antigens and to interpret shifts in flow cytometry data resulting from alterations in gene expression. Flow cytometry is used to obtain estimates for the distribution of fluorescent ligands bound to cell surface receptors throughout a cell sample. The equipment used provides light scattering parameters and also cell staining data in the form of dot plots and histograms of fluorescence intensities and the frequency of occurrence of particular fluorescence intensities. It is then assumed that fluorescence intensity is proportional to the number of labelled ligands bound to surface receptors. In this paper we present an outline of a statistical theory to account for the stretching and translation of such flow cytometry profiles which occur either as a result of alterations in gene expression, or from changing the sub-saturating concentration of fluorescent-labelled monoclonal antibodies or lectins used to stain the cells. We describe how the theory has been incorporated into two programs CSAFIT (cell surface antigen fit) and MAKCSA (make data to test CSAFIT). The program CSAFIT can be used to estimate two parameters, alpha and beta, by constrained non-linear regression analysis of the flow cytometry profiles. If the shift results from changes in the concentration of a staining agent then the estimates alpha and beta calculated by CSAFIT are functions of the ligand concentration, the ligand type and the cell line characteristics. They quantify the stretch and translation events that are encountered in flow cytometry. So when the parameter estimates alpha and beta are then further analysed as functions of ligand concentration, estimates for the average association constant K for the binding-site/ligand interaction can be obtained. This paper describes details of the development of programs CSAFIT and MAKCSA. We also discuss the distribution of parameter estimates calculated by CSAFIT and the overall performance of CSAFIT as assessed by simulation studies using data generated by MAKCSA.


Immunoglobulin VH usage analysis by fluorescent in situ hybridization and flow cytometry. We have devised a flow cytometry-based fluorescent in situ hybridization assay that permits analysis of gene expression in a large number of single cells. In this technique, fixed and permeabilized cells are incubated with biotinylated single-stranded RNA probes and by means of a fluorescently labelled second-step reagent, the cells are analyzed by flow cytometry. This is a rapid and simple method that allows all of the steps in the procedure to be performed on cells in suspension. Using this approach, we demonstrate here that immunoglobulin heavy chain variable region (VH) gene expression can be analyzed among individual cells using particular VH family-specific probes. This technique has a high degree of accuracy (greater than 97%) in detecting the fraction of cells expressing a specific message in a population and is sensitive enough to detect immunoglobulin message in LPS activated B cells. The technique has been applied successfully to monitor gene expression in homogeneous and heterogeneous populations. It also allows concurrent analysis of cell surface proteins and gene expression through two-color flow cytometry. This method of monitoring gene expression in individual cells may have a number of applications in immunology and cell biology.


PROBIT: weighted probit regression analysis for estimation of biological activity. PROBIT estimates biologic activity by weighted probit regression analysis and comparison with a known standard. The program is intended to speed up calculation of the concentration of cytokines in large numbers of cell supernatants. Data are input from either sequential or free-form text files created with a spreadsheet or text editor. This allows transfer of data from a beta-counter equipped with a suitable terminal without retyping. Results are displayed and/or printed as relative potency (% of standard) and 50% effective dose (ED50).


Calculation of antibody and antigen concentrations from ELISA data using a graphical method. A graphical method for determining the concentration of either the antibody or the antigen from ELISA data is presented in the form of a GWBASIC program. In the program, ELISAEQ, optical densities (OD) obtained from a 96-well ELISA plate can be input either directly by interfacing a microplate reader to the computer or manually. The program uses standard sample data, and selects the semilogarithmic linear range. Over this range, a least-squares method is used to determine the concentrations of interest. In addition, a hyperbolic interpolation formula is derived over the entire range for estimating the antibody or antigen concentration of the unknown samples whose OD is beyond the linear range.


A comparison of commercially available adjuvants for use in research. We evaluated an adjuvant, TiterMax, as an alternative to complete Freund's adjuvant (CFA) for producing antisera in animals. TiterMax, consists of a microparticulate stabilized water-in-oil emulsion of a metabolizable oil, squalene, with the adjuvant block copolymer CRL89-41. This paper reports two evaluations of TiterMax versus CFA and other commercially available adjuvants. In the first study, mice were immunized with a hapten, trinitrophenol, conjugated to hen egg albumin (TNP-HEA) in one of several adjuvants: TiterMax, CFA, Adjuvax, Ribi adjuvant system (RAS), Alhydrogel or Lipovant. TiterMax induced higher longer lasting titers with fewer injections than any of the other adjuvants. The magnitude of the response to TNP varied with species and route of immunization. In the second study, CFA, TiterMax, Adjuvax and RAS were compared in rabbits, mice and goats. Animals were immunized with luteinizing hormone-releasing hormone (LHRH) conjugated to BSA in each adjuvant using comparable protocols. TiterMax induced titers against the peptide equivalent to CFA in all three species. The inflammatory responses induced by TiterMax were mild and transient compared with those induced by CFA. These data suggest that TiterMax is an effective alternative to CFA in many situations.


Apoptosis in interleukin-3-dependent haemopoietic cells. Quantification by two flow cytometric methods. The murine haemopoietic cell line, BAF3, undergoes apoptosis when the growth factor IL-3 is withdrawn. Two flow cytometric methods for quantifying the apoptotic cells are described. Cell sorting followed by DNA gel electrophoresis, and both light and electron microscopy have been used to identify the apoptotic cells. In the first method the cells are fixed in ethanol, stained with propidium iodide and a DNA histogram recorded. The apoptotic cells give a 'sub-G1' peak. In the second method unfixed cells are incubated with the bis-benzimidazole, Hoechst 33342. The apoptotic cells take up this dye more rapidly. In this latter method, the non-viable cells can also be enumerated by addition of propidium iodide. The value of the method has been demonstrated in a brief study of the effects of a panel of cytokines on growth and apoptosis.


Detection of lipopolysaccharide (LPS) and identification of its serotype by an enzyme-linked immunosorbent assay (ELISA) using poly-L-lysine. A new solid-phase enzyme-linked immunosorbent assay (ELISA) was developed for detection of LPS and identification of its serotype with antisera. Since LPS binds poorly to polystyrene microplates, precoating with poly-L-lysine was used before coating LPS on the surface of microplates. The small amount of LPS in complex mixtures (i.e., less than 1 microgram/ml) could be detectable in ELISA. Use of poly-L-lysine with high molecular weight (MW) provided a higher sensitivity than poly-L-lysine with low MW. Precoating with polymyxin B, or poly-L-histidine was less effective in the sensitivity than precoating with poly-L-lysine, but it was still better than no precoating. The newly developed ELISA technique could be also applied for detection of anti-LPS antibodies in sera or for screening of monoclonal anti-LPS antibody.


In vivo immunosorption--a method for the examination of hormone heterogeneity at low plasma concentrations. Application to progastrin and its products. In order to investigate whether antibody desorption followed by chromatography is useful for the study of ligand heterogeneity in plasma, endogenous plasma gastrins were released from immunoglobulins by denaturation of antisera from 11 rabbits immunized against different fragments of human progastrin. The molecular nature of the in vivo immunosorbed plasma gastrins was characterized by gel chromatography monitored by a library of sequence-specific radioimmunoassays before and after enzyme cleavage. Subsequently, the plasma gastrins were compared with tissue gastrins in extracts of rabbit antral mucosa. The results show that carboxyamidated gastrins and their immediate glycine-extended precursors circulate in rabbit plasma. The carboxyamidated gastrins eluted as gastrin-34 and gastrin-17, both in sulfated and non-sulfated form. Correspondingly, glycine-extended gastrin-34 and gastrin-17 also occurred in plasma. The results confirm that the plasma of immunized animals contains substantial quantities of the corresponding endogenous ligands bound to specific antibodies. This in vivo immunosorption phenomenon can be used to study the molecular heterogeneity of hormones and their precursors, when they circulate in concentrations which are otherwise too low to permit examination of their molecular nature.


An improved and rapid method for the isolation of rat lymph node or spleen T lymphocytes for T cell proliferation assays. To detect and compare the capacity of antigen presenting cells to present antigen in a T cell proliferation assay, it is necessary to obtain a pure population of antigen-primed T cells that gives low background proliferative responses. Therefore in this paper we present a newly developed isolation method of antigen-primed T lymphocytes from rat spleen or lymph nodes. This method uses a nylon wool column to deplete most of the adherent cells and B cells, followed by an indirect elimination method with magnetic beads to remove contaminating Ia-positive cells. We compared this method with two commonly used isolation methods, namely a 1.5 h adherence step, followed by a nylon wool column and a Sephadex G-10 column and a 1.5 h adherence step followed by a passage through two consecutive columns of Sephadex G-10. The best T cell enrichment (98% OX-19/52-positive cells) was achieved with the newly developed method, in which the contamination of Ia-positive cells, predominantly B cells and dendritic cells (DC), was diminished to less than 2%. The background response of this population was low and differed significantly with the common methods. Antigen-specific T cell responses induced by splenic DC, expressed as stimulation index, gave very specific responses and showed a steep rise with increasing DC concentrations compared to the common methods. Therefore we conclude that we developed an improved, rapid and reproducible method for the isolation of rat spleen or lymph node T lymphocytes suitable for T cell proliferation assays.


A simple standardised protocol for the production of monoclonal antibodies against viral and bacterial antigens. A simple standardised protocol for making monoclonal antibodies against a range of human bacteria and viruses is described. The protocol was designed to reduce the number of steps to a minimum. A one step footpad immunisation was followed by the fusion schedule 10-15 days later. A vital step in the technique was the use of the immunised mouse's spleen to provide a feeder layer post fusion. This simplified the protocol and more importantly greatly accelerated the growth of the hybridomas produced. Immunisation, fusion and clonal expansion of specific antibody secreting hybridomas was complete within 5 weeks. The percentage of hybridomas secreting specific antibody ranged from 6% to 28%, the majority of which were of the IgG isotypes. The method was economical in the use of tissue culture medium and simple to perform.


A cleavable biotinylating agent and its use in protein electroblots. A direct method for the synthesis of N-biotinyl penicillamine is described. It has been shown to be a convenient biotinylating agent for antibodies which have been previously coupled with SPDP. The biotinylated antibodies can be used to detect antigens on protein electroblots using 125I-labelled streptavidin and radioautography. The biotin and its attached streptavidin and radiolabel can be removed under mild conditions and the blot reprobed with a different antibody using an identical protocol.


Determination of IgE antibodies to the benzyl penicilloyl determinant. A comparison between poly-L-lysine and human serum albumin as carriers. The influence of two different carriers, poly-L-lysine (PLL) and human serum albumin (HSA) in the binding of specific IgE antibodies to the benzyl penicilloyl hapten (BPO) was determined in a solid-phase assay. Serum samples from patients with a history of immediate reaction to penicillin and which had shown the presence of IgE antibodies to BPO were used. Benzyl penicilloyl derivatized cellulose discs were prepared using PLL of different molecular weight and HSA as carriers. These were treated with different molar ratios of benzyl penicillin. These reagents were compared for uptake of BPO-specific IgE using a pool of sera in a radioallergosorbent test (RAST) type assay. Two PLL systems and two HSA systems were finally compared using 26 individual sera. RAST values were compared by Kruskal-Wallis and Wilcoxon tests. The relationships between the four different assays were evaluated by determining Pearson correlation coefficients and the concordance by determining intraclass correlation coefficients (ICC). Analysis of means by the Wilcoxon test revealed significant differences (P less than 0.01) only when the different carrier assays were compared. The correlation coefficients between all the assays were significant (P less than 0.0001), but the ICC was low when the different carrier assays were compared. These results indicate that the nature of the carriers studied (PLL and HSA) influences the capacity for binding IgE antibodies in the RAST procedure. The differences observed indicate that one conjugate cannot be substituted for the other in the determination of IgE antibodies to BPO and that BPO-PLL is preferable.


Inspiratory spirogram in emphysema.

Inspiratory spirogram in emphysema. Bronchodilator therapy on inspiratory indices has not been documented so far. Reduction in inspiratory airflow resistance following inhalation of bronchodilator giving the most consistent and significant changes in emphysematous patients has been demonstrated in the present study.


Jaundice in pregnancy: a clinical study. In a study on jaundice during pregnancy it was observed that the incidence of jaundice in pregnancy was 1 in 429. Majority of the cases were primigravida, 70% of cases were between 20 and 30 years of age, 60% of the patients were unbooked, 25% of cases were with severe degree of jaundice in respect to serum bilirubin level and 70% cases were with anaemia. There were preponderance of cases in 3rd trimester. Majority of the cases (60%) had jaundice-delivery interval of above 4 weeks. Among the causes virus hepatitis was commonest one (80%). Maternal mortality was recorded as 1 in 20 cases and perinatal mortality was 7 in 17 births (41%).


Study of circle system without absorber under controlled ventilation with limited fresh gas flow. A biphasic controlled randomised study was conducted to evaluate the differences in fresh gas flow with alterations in different anaesthetic parameters, on 15 adult female patients of American Society of Anesthetists grade I or II, who underwent elective gynaecological operations with identical premedication and anaesthetic technique in which absorber was by-passed. Intermittent positive pressure ventilation was maintained with a tidal volume of 10 ml/kg and fixed respiratory frequency of 16/minute with inspiratory:expiratory ratio of 1:2. N2O (67%) in O2, a non-depolarising muscle relaxant and a narcotic analgesic were used to maintain the anaesthesia, muscle relaxation and analgesia respectively. Phase I and II of the study were characterised by fresh gas flow of 81/minute and 5.61/minute respectively during steady state and data of both phases were collected every 30 seconds for 5 minutes. Arterial blood samples for arterial CO2 and O2 tension studies were taken towards the end of 5-minute period. In phase I a mean fractional concentration of end tidal CO2 of 3.43% +/- 0.42 (p less than 0.01) and a mean arterial tension of CO2 of 24.06 mm of Hg +/- 4.07 were obtained. In phase II mean fractional concentration of end tidal CO2 3.81% +/- 0.39 (p less than 0.01) and mean arterial O2 of 28 mm Hg +/- 2.32 were obtained. Mean arterial blood O2 saturation was above 99% and mean arterial O2 tension was above 160 mmHg in both the phases. The results were statistically significant.


Maternal mortality in a subdivisional hospital of eastern Himalayan region. This study was conducted in a subdivisional hospital of eastern Himalayan region among 5,273 pregnant women over a period of 8 years. There were 29 deaths, the maternal mortality rate was 55 per 10,000. Septic abortion was encountered in 4 among them. Direct obstetric cause was responsible in 72.41% of cases and indirect cause in 27.59% cases. Sepsis, both puerperal and postabortal resulted in 24.14% followed by postpartum haemorrhage in 20.69%. Two of these cases were associated with inversion of the uterus. Preeclampsia caused 10.34% and eclampsia 6.9% of the deaths. Among the indirect causes severe anaemia and pulmonary tuberculosis accounted for 10.34% and 6.9% respectively. Infective hepatitis was the cause in 6.9% cases. Only 17% of the cases were booked and the rest were unbooked. Majority of the cases (62.07%) belonged to the age group of 20-30 years. Primigravida constituted 41.38% of the cases.


Effect of fenfluramine on serum T3, T4 and TSH levels in obesity. A prospective, randomised case-control trial of an anorectic drug, fenfluramine was conducted on 30 patients of simple obesity. The study revealed that the drug was well tolerated, non-toxic and effective in reducing the body weight and normalising the thyroid profile. Reduction in body weight, rise in serum thyroxine (T4) and fall in serum triiodothyronine (T3) was highly significant (p less than 0.01) in drug treated group as compared to controls (p less than 0.05) after 12 weeks of therapy. There was also fall in serum thyroid stimulating hormone (TSH) levels but without any statistical significance.


System functionality and physicochemical model of fentanyl transdermal system. Fentanyl is an opioid traditionally administered by infusion or injection and more recently in a rate-controlled transdermal dosage form. This system is a four-layer laminate on a protective liner. A backing layer seals and protects the drug reservoir, the source for continuous delivery of fentanyl. A membrane controls the release rate of fentanyl from the system. An adhesive layer attaches the system to skin and releases an initial loading dose of fentanyl. The rate of fentanyl delivery through skin is determined by the system and the skin at the application site. The release rate from the system is approximated by Fick's first law of diffusion and is controlled by the rate-controlling membrane. A complete simulation model that combines both in vitro release data and the pharmacokinetic model has been developed and used to show the influence of various physiologic and system variables on serum fentanyl concentrations.