H. S. Lawrence

An Improved Method for Post-PCR Purification for mtDNA Sequence Analysis

Mitochondrial DNA (mtDNA) analysis of forensic samples typically is performed when the quantity and quality of DNA are insufficient for nuclear DNA analysis or when maternal relatives may be the only reference source. Many of the steps required in the analytical process are both lengthy and labor intensive. Therefore, improvements in the process that reduce labor without compromising the quality of the data are desirable. The current procedure requires purification of the amplicons by centrifugal filtration after PCR and prior to cycle sequencing. Because this method requires several manipulations to perform, alternate cleanup procedures were investigated. These include the use of 1) Qiagen QlAquick PCR Purification columns, 2) Concert Rapid PCR Purification columns, and 3) ExoSAP-IT reagent. When the yield of purified amplicons, quality of the sequence profile, and ease of assay were evaluated, the use of ExoSAP-IT reagent for post-amplification purification was chosen to replace the filtration method.

Treatment of cryptosporidiosis with oral bovine transfer factor

Cryptosporidia are intestinal protozoans long known to cause diarrhea in humans, especially those with acquired immune deficiency syndrome (AIDS). When transfer factor prepared from calves which possessed delayed-type hypersensitivity to Eimeria bovis was given to nonimmune calves and mice it conferred protection against clinical infection (coccidiosis). Recent studies with oral bovine transfer factor have shown that it can confer cell-mediated immunity to humans. Based on these findings we decided to treat eight AIDS patients suffering from Cryptosporidium-associated diarrhea with transfer factor prepared from calves immune to Cryptosporidium. Prior to treatment with transfer factor, three patients had been treated with spiramycin, one patient with alpha-difluoromethylornithine (DFMO), and one patient with furazolidone for greater than 1 month without clinical or laboratory improvement. Following administration of transfer factor, five or eight patients exhibited a decrease in the number of bowel movements and the development of formed stools. Cryptosporidium was eradicated from the stools of four patients but two of these patients subsequently relapsed and one patient continued to have diarrhea despite the absence of Cryptosporidium in the stool. One patient has been free of diarrhea and Cryptosporidium for 2 years after discontinuation of transfer factor therapy.

Preparation and properties of cloning inhibitory factor. I. Inhibition of HeLa cell cloning by stimulated lymphocytes and their culture supernatants.

Abstract Lymphocytes stimulated by either phytohemagglutinin or tuberculin inhibit the cloning of HeLa cells. Cell-free supernatants prepared from such cultures of activated lymphocytes produced similar inhibition, whereas supernatants from unstimulated lymphocytes did not. The cloning inhibitory factor (CIF) produced by activated lymphocytes was nondialyzable and inactivated at 56 °C for 30 minutes. CIF did not produce cytolysis of cloned HeLa cells; time-lapse cinematography instead disclosed an additional, normally timed mitosis. Subsequent divisions, however, were markedly delayed. CIF did not depend for its activity on depletion of an essential nutrient from the HeLa medium.

Erythrocytes in human transplantation: effects of pretreatment with ABO group-specific antigens

Erythrocyte group antigens A and B can act as potent and group-specific transplantation antigens in man. ABO group-incompatible recipients pretreated with such antigens have rejected skin allografts obtained from donors incompatible for the same antigens in an accelerated (4-5 days) or white graft manner. Skin grafts applied to the same recipients from ABO-compatible donors were accorded first-set survival times. Intact erythrocyte suspensions and antigens isolated from hog (A substance) and horse (B substance) stomachs, were equally capable of inducing this type of allograft sensitivity. The latter observation broadens the spectrum of heterologous antigens capable of inducing allograft sensitivity in the mammalian host and provides a readily available, heat-stable, and water-soluble source of antigens for further studies of allograft rejection mechanisms in man.

Effects of prepubertal stress on subsequent ACTH response to novel stress and CRH in male vs female rats

The effects of long-term chronic stress during prepubertal periods of growth and development on an organisms ability to release ACTH during future episodes of an acute novel stress and in response to exogenous CRH were examined. Following a 6-week stress period, in which prepubertal male and female WKY rats were subjected to three different and randomly given stress paradigms (heat, noise and immobilization) at various times of the day (in order to prevent adaptation to stress), chronically stressed male rats were far less able to respond to CRH plus a novel ether stress than were their male controls or their female counterparts. Although baseline ACTH levels were similar in both male and female control and experimental rats, when subjected to a subsequent acute ether stress, the differences in ACTH response between controls and experimentals as well as between males and females were significant. ACTH response to stressors was significantly blunted in both male and female experimental rats compared to their controls, but the male response was significantly lower than that of the females. These results suggest that prepubertal chronic stress may permanently alter an organisms ability to release ACTH, even when subjected to a novel and traumatic ether stress, and that males may be much more susceptible than females to prepubertal stress. Long-term stress, therefore, if experienced during critical developmental periods such as preadolescence, can permanently damage the stress response mechanism and cause other, more serious physiological disorders.

THE RELATIONSHIP OF IN VITRO LYMPHOCYTE COMPATIBILITY TO HOMOGRAFT SENSITIVITY IN MAN

In another paper’ in this monograph, we have presented evidence that cultured human peripheral blood lymphocytes are immunologically capable and show immunological specificity. As outlined in that paper, their degree of response to specific and nonspecific stimuli can be quantitated by counting the number of cells which have enlarged in culture and those cells undergoing mitosis. Wilson2 has shown that if a n animal is immunized with a skin graft from an unrelated animal, thoracic duct lymphocytes from the recipient kill cultured renal cells derived from an animal of the donor’s strain. These lymphocytes will not attack renal cells from other strains known to have markedly different histocompatibility antigens, demonstrating the specificity of this cytotoxic effect. Many investigators have used lymphocytes to sensitize individuals, producing accelerated rejection of the skin graft from the donor of the lymphocytes to the immunized individual. This shows that the lymphocyte contains histocompatibility antigens and is capable of producing an immune reaction to these. Therefore, the lymphocyte appears to be representative of both the donor’s and the recipient’s contribution in transplantation. Brent and Medawar? have used lymphocytes injected subcutaneously to test for genetically determined histocompatibility differences between various animals. They have found no reaction to injected lymphocytes transferred within an inbred strain. This correlates with the finding by numerous investigators that transplants from one identical twin to the other are not rejected. These observations provide a basis for the assumption that differences in histocompatibility antigens are genetically determined. Bain et al.* have shown that if peripheral blood lymphocytes from two unrelated individuals are mixed in vitro, there is an increase in the incorporation of radioactive thymidine into DNA. Monozygous twins showed no such response, while among a series of dizygous twins, two pairs were found who also did not react with each other. We have done similar studies on lymphocyte mixtures. However, our method of quantitation depends on counting the percentage of cells which have become enlarged in culture, and adding to these the percentage of cells in mitosis, since these latter derive from enlarged cells. The details of the culture methods are described e l ~ e w h e r e . ~ A time curve of this response (FIGURE 1) demonstrates that the peak of observable reaction occurs between seven and eight days. The degree of this reaction varies from no increase over the spontaneous rate of enlargement and mitosis (approximately five per cent) in identical twins, to a response of 85 per cent observed in two individuals from widely divergent ethnic backgrounds. As a rule, blood relatives show less of a response. than unrelated

In vitro studies on transplantation immunity. I. MIF production by sensitive lymphocytes in mice.

Abstract Sensitized lymphocytes from mice immunized with skin homografts produce migration inhibitory factor upon incubation with lymphocytes (antigen) from the sensitizing strain. The MIF is produced within 14 hr following incubation of sensitized lymphocytes and antigen. In this reaction, antigenic specificity is a prerequisite for MIF production; however, the action of MIF transcends the strain barrier. Also, MIF produced in homograft reactions in mice inhibited the migration of peritoneal cells from normal guinea pigs. Finally, lymphocytes from mice bearing skin homografts do not develop the capacity to produce MIF prior to the rejection of the sensitizing skin grafts.

ANTIGEN-SPECIFIC SUPPRESSOR FACTOR IN HUMAN LEUKOCYTE DIALYSATES: A PRODUCT OF TS CELLS WHICH BINDS TO ANTI-V REGION AND ANTI-Ia REGION ANTIBODIES

Publisher Summary This chapter describes experiments in which an antigen-binding inducer factor and an antibody-binding suppressor factor were isolated and characterized, each present in the same human leukocyte dialysate (DLE) fraction (>3500 3500

SERUM ANTIBODY RESPONSE TO TISSUE TRANSPLANTATION ANTIGENS IN MAN

There is general agreement that the rejection of homologous tissue transplants is achieved through a n immunological response undertaken by the h0st.l Some of the mechanisms invoked in homograft reactions appear to bear a similarity to responses observed in the delayed type of hypersensitivity.2 However, the possibility also exists that some form of serum antibody response is associated with homograft rejection in certain animal species3 In man, however, the one fully documented report of a serum antibody response to skin homograftsZ was based on studies of a severely burned child, in whom many factors other than the applied grafts may have been operative, and, on the results of Van Rood, in subjects also sensitized by injections of blood ~ l a t e l e t s . ~ Colombani et al. have been unable to demonstrate a leukoagglutinating serum antibody response in normal skin homograft recipients,6 and the attempt to transfer skin homograft sensitivity with serum obtained from hypersensitized human white graft reactors has also been un~uccessful .~ Thus, within the limitations of experimental models used, specific serum antibody response to skin homograft rejection in man has been a rather rare finding. The systematic study of human subjects sensitized to skin homografts in our laboratories by the injection of various preparations of human blood leukocytes afforded a further opportunity to study this problem at various stages following sensitization and homograft rejection. For this purpose, each individual studied in the course of attempts to localize human transplantation antigens present in blood leukocytes was also investigated from this standpoint.

Preparation and properties of cloning inhibitory factor: II. Factors affecting its production and assay

Abstract Cloning Inhibition Factor (CIF), an activity present in PHA or antigen stimulated lymphocyte culture supernatants, inhibited the cloning of HeLa cells when diluted 1:9 in HeLa culture medium. CIF was not detectable at 8 hr, was maximal at 24–48 hr, and declined with longer periods of lymphocyte culture. CIF production increased with lymphocyte concentration up to 1–2 × 10 6 lymphs/ml but plateaued at higher concentrations. At lower lymphocyte concentrations, more CIF activity was present when lymphocytes were cultured in 5% rather than 12% serum. PPD elicited similar CIF activity from either highly purified or unpurified lymphocytes. CIF activity was independent of HeLa medium serum concentration. It remained stable for 3–6 months at −20 °C, but was inactivated by heating at 56 °C for 30 min. At a 1:9 dilution CIF was not cytocidal but produced cytopathic changes. CIF shares many properties with, and may be identical to, Proliferation Inhibitory Factor.