The factor of immunization in the rat. The effect of allogeneic immunization on graft-versus-host activity.

Using a popliteal lymph node weight assay the graft-versus-host activity of lymphocytes from donors immunized with allogeneic tissue has been assayed by comparison with that of lymphocytes from nonimmune donors. When the donors were immunized against weak histocompatibility antigens (non-AgB) the specific GVH activity of its lymphocytes was increased. This increase was greater if spleen cells rather than thoracic duct lymphocytes were the source of the donor cells used for assay. The increase in GVH activity was also greater if the standard immunization procedure of two successive skin allografts was followed by three boosting injections of allogeneic lymphoid cells. When donors were immunized against strong histocompatibility antigens the specific GVH activity of the donors' lymphocytes was slightly increased, was unchanged, or was actually decreased depending on the experimental situation. In donors rendered incapable of a humoral alloantibody response by whole body X-irradiation, immunization across a strong barrier was followed by little or no increase in the specific GVH activity of TDL. In the rat, as in other species, the increase in GVH activity after immunization is inversely proportional to the strength of the antigenic barrier involved.

Early cellular events in a systemic graft-vs.-host reaction. I. The migration of responding and nonresponding donor lymphocytes.

A systemic graft-vs.-host (GVH) reaction was initiated by the intravenous injection of parental strain thoracic duct lymphocytes (TDL) into irradiated F1 hybrid recipients with in-dwelling thoracic duct cannulae. The migration of the donor lymphocytes was followed by labeling them in vitro with either [3H] or [14C]uridine and measuring radioactivity by scintillation counting of the spleen and lymph nodes of the recipients removed 24 h after injection and in TDL collected throughout this period. The localization of labeled cells was always compared to that of a reference population of nonreactive lymphocytes, e.g. F1 hybrid, labeled with the alternative isotope (Fig. 1). A consistent surplus of the reactive label was found in the spleen which was balanced by a deficit of the reactive label in TDL; lymph nodes gave intermediate values. The same distribution pattern was noted when the reference population was a specifically unresponsive population of the parental strain. This differential distribution depends on recognition of the recipient's Ag-B antigens because when normal lymphocytes were injected together with specifically unresponsive lymphocytes into a "third party" F1 hybrid (against which both populations were reactive) there was no surplus of the normal cells in the spleen and no deficit in the lymph. Moreover in an Ag-B identical strain combination there was no detectable difference in the distribution of reactive and nonreactive populations. The distribution of a labeled reaction population can be accounted for if a substantial minority of cells are immobilized in the spleen and lymph nodes as a consequence of antigen recognition (Fig. 3). When the donor cells in the spleen were assayed 24 h after injection there was paradoxically a slight reduction in their specific GVH activity, which is at least partly because they are under-represented in a single cell suspension. The size of the splenic surplus (23%) and the thoracic duct deficit (12%) suggested that the minority of nonimmune lymphocytes which recognize each Ag-B complex carry 12% of the radioactive label in the original population. It is argued that this provides a near estimate of the frequency of T lymphocytes which can recognize each Ag-B antigenic complex.

Early cellular events in a systemic graft-vs.-host reaction. II. Autoradiographic estimates of the frequency of donor lymphocytes which respond to each Ag-B-determined antigenic complex.

A graft-vs.-host (GVH) reaction was initiated by the intravenous injection of parental strain (AO) lymphocytes into irradiated (AO times HO)F1 or (AO times DA)F1 hybrids. The proportion of donor T cells which had responded to the F1 hybrid antigens within 24 h was estimated by two methods. (a) Donor lymphocytes were labeled with [3H]uridine in vitro before injection. The proportion of labeled cells which had morphologically transformed in the recipient's spleen was 17-19%. In both series of experiments syngeneic transfers were performed in which case the proportion of transformed cells was 1-2.4%. A similar low proportion was found after parental to F1 transfer in a non-Ag-B strain combination. These figures were used to calculate the frequency of responding cells in the injected population given three additional pieces of information: (a) the extent of selection in the spleen which transformed the estimate to 4.5%-6.0% responders; (b) division of donor cells was shown to be negligible under the conditions of the experiment; and (c) the nonspecific recruitment of lymphocytes was shown to be negligible. A speculative model of antigen recognition by T cells which accounts for the high proportion of responders is outlined.

The specific selection of recirculating lymphocytes by antigen in normal and preimmunized rats.

Thoracic duct lymphocytes (TDL) from normal rats will restore a primary antibody response to sheep erythrocytes (SRBC) in irradiated recipients and cause a graft-versus-host reaction in F(1) hybrid rats; lymphocytes from rats immunized with either tetanus toxoid or dinitrophenylated bovine gamma globulin (DNP BGG) will generate specific antibody after cell transfer and challenge. The ability of TDL to mediate each of these responses is severely depressed by giving a single intravenous dose of the specific antigen shortly before cannulation of the thoracic duct, although the lymphocyte donors themselves respond normally. The injection of antigen does not decrease the output of lymphocytes in the thoracic duct and the effect is specific for the antigen injected. The findings are most readily accounted for by assuming that small subpopulations of specific lymphocytes are selected from the recirculating pool by antigen which has localized in lymphoid tissue. The observation that passive antibody abolishes selection by SRBC supports this interpretation. The strong selection exerted by a subcutaneous injection of SRBC in Freund's complete adjuvant, which induces delayed hypersensitivity but little early antibody, suggests that a common cell type may be involved in the induction of both delayed hypersensitivity and antibody formation. The anti-DNP antibody response generated by TDL from rats immunized with DNP BGG was abolished by a selecting injection of the homologous conjugate. The response was depressed to a smaller degree by injections of either BGG or dinitrophenylated human serum albumin, suggesting that carrier-specific (T) and hapten-specific (B) lymphocytes could be separately selected from the recirculating pool. The regional selection of recirculating lymphocytes by antigen may explain a number of phenomena in which the prior injection of antigen has been found to inhibit a subsequent immune response.

Reasons for HIV antibody test refusal in a heterosexual sexually transmitted disease clinic population.

OBJECTIVE: To evaluate acceptance of confidential HIV antibody testing and reasons for test refusal among heterosexual clients of Los Angeles County sexually transmitted disease (STD) clinics. METHODS: From January 1993 through June 1994, all blood specimens routinely collected for syphilis serology were tested blindly for HIV antibody at seven STD clinics. Patients were counseled and offered a confidential HIV test. Rate of refusal of confidential testing and primary reason for test refusal were examined by demographic group and HIV serostatus, as determined in the blinded survey, for all heterosexual clients. RESULTS: Of 20,125 persons offered confidential testing, 35.6% refused the test. Test refusal was higher among men (38.7%) than women [31.1%; adjusted odds ratio (OR), 1.4; 95% confidence interval (CI), 1.3-1.4] and among blacks (38.6%) than whites (28.6%; adjusted OR, 1.7; 95% CI, 1.5-2.0). The most common reason for refusal was 'already know my HIV status' (40.6%), followed by 'don't want to know' (23.9%), and 'not at risk' (19.4%). Confidentiality concerns were cited as the primary reason for refusal by 2.2%. Among the 180 (0.9%) persons who tested positive in the blinded survey, 99 (55.0%) refused the confidential test. Of the 44 seropositive persons who refused the confidential test because they "already knew their HIV status', 29 (65.9%) reported their previous test to be negative. CONCLUSIONS: Efforts are needed to increase acceptance of confidential HIV testing in this heterosexual population and should (1) include a client-centered counseling approach that facilitates accurate self-assessment of risk and addresses the misperception that a prior negative test result implies an absence of risk, and (2) highlight the potential benefits of early intervention medical and psychosocial services.

The selective adherence of lymphoblasts to antigenic cell monolayers. A method for determining the specificity of lymphocytes proliferating in response to histocompatibility antigens.

The specificity and intensity of the immune response of rat lymph nodes draining a skin allograft were examined by exploiting a monolayer of donor-type thoracic duct lymphocytes as an immunoabsorbent. Stable monolayers were produced by attaching lymphocytes from different strains of rat to Petri dishes pretreated with poly-L-lysine. The responding lymph node cells were labelled in vitro with [3H]Thymidine, incubated on the monolayer and mechanically separated into non-adherent and adherent fractions. The radioactivity associated with the adherent fraction was 7--8 times greater when the monolayer displayed the immunizing major histocompatibility antigens than when syngeneic or 'third party' monolayers were used. The non-specific adherence to syngeneic monolayers was low and consistent. Immunization to minor histocompatibility antigens may also be studied by this method.

Factors influencing the fate of 111indium-labelled lymphocytes after transfer to syngeneic rats.

Thoracic duct lymphocytes were labelled in vitro with 111indium-oxine or 111indium-acetylacetone in order to follow their migration after i.v. injection into syngeneic rats. Under certain conditions both preparations produced results with quantitatively confirmed data obtained by other approaches to the physiological pattern of lymphocyte recirculation. However, three significant difficulties were identified: (1) chemical toxicity by minor contaminants of the preparation; (2) radiation damage indicated by a progressive impairment of the recovery of radioactivity from lymph nodes. A labelling concentration of 20 microCi/10(8) cells was the highest compatible with survival of most lymphocytes for 24 h in vivo as confirmed by autoradiography; (3) rapid loss of 111In in vivo found at labelling concentrations below 1 microCi/10(8) cells. By one week after the injection of lymphocytes labelled at 20 Micro/Ci/10(8) cells most of the 111In had been transferred from lymphocytes to non-recirculating radioresistant cells within the spleen and lymph nodes.

Major histocompatibility complex control of NK-related allogeneic lymphocyte cytotoxicity in rats. The contributions of strong and medial transplantation antigens.

Allogeneic lymphocyte cytotoxicity (ALC) describes the elimination of allogeneic lymphocytes in vivo by an NK-related activity. There is evidence that ALC is demonstrable between donor and recipient when these are incompatible at MHC gene loci alone. Since ALC is a property of T cell-deficient nude rats, the role of the MHC in this rejection process needs further study. We have determined the contribution of the MHC to ALC using congenic and recombinant rats. In our analysis we have assumed that ALC involves the recognition of classic alloantigens by clonally distributed effector cells as for other examples of transplant rejection, although this is not yet proved. Strong ALC was measured between congenic rats that differed for MHC genes only. Non-MHC incompatibility alone did not elicit ALC. In the presence of MHC incompatibility the strength of ALC generated in a recipient was dependent on non-MHC genes. The PVG background generated high ALC responses whereas ALC was not measured in the DA rat. However ALC was measured in the congenic PVG-RT1avl (DA) rat. The contributions of classic class I (RT1.A), class II (RT1.B/D), and medial transplantation (RT1.C) regions of the rat MHC were determined by comparing different recombinant donors into the same recipient strain. Single region differences alone in any of these three MHC regions did not elicit full ALC. In two sets of transfers a combination of RT1.B/D and RT1.C region incompatibility was sufficient to generate a full allogeneic response. It can be concluded that the controlling element for allogeneic lymphocyte cytotoxicity is in the RT1.B/D-RT1.C region of the rat MHC.

A comparison of immune responses against AG-B and non-AG-B antigens, presented alone or together.

By exploiting congenic rat strains (HO.B2 and PVG/c) cell-mediated immune responses against Ag-B antigens alone were measured and compared with responses against (1) non-Ag-B antigens and (2) Ag-B and non-Ag-B antigens in combination. It was confirmed that multiple non-Ag-B antigens provoke prompt first-set skin graft rejections, but are much weaker than Ag-B antigens in stimulating both graft-versus-host (GVH) and cytotoxic activity. No evidence of synergistic interaction was found between anti-Ag-B and anti-non-Ag-B responses either by GVH assay or in the generation of cytotoxic cells. Specific partitioning of cytotoxic cells on antigenic monolayers suggested that cytotoxic cells on antigenic monolayers suggested that cytotoxicity is predominantly directed against Ag-B antigens. The measurements of GVH activity consolidate previous work, which suggested that 4.5 to 12% of nonimmune T cells can respond to each Ag-B determined antigenic complex and eliminate the possibility that most of these cells were responding to non-Ag-B antigens. Two principles for measuring GVH activity were compared: (1) 3H-thymidine incorporation into donor lymphocytes at 24 hr after transfer to irradiated F1 hybrid recipients and (2) the popliteal lymph node assay, which depends on a secondary phase of host cell proliferation.

A probability-based approach for predicting HIV infection in a low prevalent population of injection drug users.

This article proposes a method for estimating HIV risk in low-HIV-prevalent populations. Allard's risk probability model was used to compute individual risk scores. Based on a sample of 3854 injection drug users (IDUs) who were confidentially tested for HIV at five methadone treatment clinics in Los Angeles County, the following self-reported risk behaviors were used to derive an individual IDU risk score: (i) frequency of injection, (ii) frequency of using uncleaned needles, (iii) number of people sharing a needle, (iv) frequency of needle sharing, and (v) type of needle sharing practice. The overall HIV prevalence for the IDU sample was 2%. The risk score was strongly associated with HIV seropositivity (chi-square = 16.1, p < 0.0001), but only one of the individual IDU risk behaviors (needle cleaning) was significantly associated with HIV seropositivity (chi-square = 10.9, P < 0.001). In addition, the risk score was strongly associated with HIV serostatus for both males and females. For females, however, none of the individual IDU risk behaviors were associated with HIV serostatus. Our findings indicate that when predicting HIV infection in a low-prevalence population, the probability-based risk score makes a statistically significant contribution over individual IDU risk behaviors.

Afferent lymph and lymph borne cells: their influence on lymph node function.

In AO rats the afferent lymphatics to the right cervical lymph nodes (LN) were interrupted and the LN were encased in silicone rubber tubes to prevent reunion of the lymphatics. At regular intervals over the next 12 weeks the following were measured in comparison with the intact contralateral LN - LN weight, influx of lymphocytes from the blood, blood flow, the incorporation of 125IUdR and the incorporation of 35S-sulphate into high endothelial venules (HEV). Systematic histological observations are also reported. One day after deafferentization lymphocyte influx was significantly reduced although blood flow was unchanged and a temporary increase in LN weight was associated with crowding of the lymphatic sinuses with small lymphocytes. The subsequent decline in lymphocyte influx was biphasic and quicker than the decline of other parameters--being undetectable by 6 weeks. Flattening of HEV and diminished secretion of 35S-sulphate was noted at 1 week and progressive degeneration and eventual disappearance of the HEV network was seen by 6-12 weeks. Doubtlessly because of lack of antigenic stimulation 125IUdR incorporation, and numbers of lymphoblasts, plasma cells and finally germinal centres were progressively reduced. The numbers of macrophages and interdigitating cells (IDC) were greatly reduced by 3 weeks and very few were present at 6 weeks probably because most or all arrive in afferent lymph and have a limited life span in the LN. At 12 weeks the LN was difficult to recognize as such since only stromal cells and occasional small lymphocytes remained. In supplementary experiments u.v. irradiation of the LN at the time of deafferentization reduced lymphocyte influx without affecting blood flow suggesting that a u.v. sensitive cell like the IDC may influence lymphocyte influx. In conclusion the involution of the deafferentized LN is partly due to the lack of antigen but progression to the complete loss of specialized structure and function is probably due to lack of other factors including non-lymphoid cells that normally arrive in afferent lymph.

Lymphocyte traffic in pregnant or oestrogen stimulated rats.

Lymphocyte migration was studied at three stages of rat pregnancy by the tracer sample principle of injecting syngeneic thoracic duct lymphocytes labelled with chromium-51 (for organ counting) or [3H]leucine (for autoradiography). Except for one abnormal embryo no entry of lymphocytes into the foetus was detected. There was a small localization of lymphocytes in the placenta which did not differ significantly between allogeneic or syngeneic mating. Pregnancy produced little or no alteration of lymphocyte traffic into the spleen or lymph nodes, including those draining the uterus. The administration of oestrogens to virgin rats increased the height of the specialized endothelium lining the post-capillary venules in the mesenteric lymph nodes but had no effect on lymphocyte traffic.

An outbreak of furunculosis among high school athletes.

Furuncles (boils) are common among teenagers; however, few outbreaks have been documented. We investigated an outbreak of furuncles that occurred among male athletes of a Kentucky high school during the 1986 to 1987 school year. The overall attack rate was 25% (31/124). The risk of developing a furuncle increased two to three times in those who had skin injury. Athletes who sustained abrasions more than twice per week (P less than 0.01), who had a cut that required bandaging (P = 0.01), or had an unspecified injury causing a missed practice or game (P = 0.04) were at increased risk. The risk of developing furunculosis did not appear to be related to contact with formites, but rather, to contact with furuncles. Although athletes shared common areas (showers, locker rooms, practice areas, the attack rates for varsity football (36%) and varsity basketball (33%) were four times greater than for nonvarsity teams (P less than 0.01). Players who had a friend with a furuncle were more than twice as likely to also have had a furuncle (P less than 0.01). Exposure to furuncles appeared to increase the risk of furunculosis independently of reported skin injury. Control and prevention should, therefore, focus on both reducing skin injury and reducing exposure to furuncles, rather than attempting to sterilize inanimate objects.

A venue-based method for sampling hard-to-reach populations.

Constructing scientifically sound samples of hard-to-reach populations, also known as hidden populations, is a challenge for many research projects. Traditional sample survey methods, such as random sampling from telephone or mailing lists, can yield low numbers of eligible respondents while non-probability sampling introduces unknown biases. The authors describe a venue-based application of time-space sampling (TSS) that addresses the challenges of accessing hard-to-reach populations. The method entails identifying days and times when the target population gathers at specific venues, constructing a sampling frame of venue, day-time units (VDTs), randomly selecting and visiting VDTs (the primary sampling units), and systematically intercepting and collecting information from consenting members of the target population. This allows researchers to construct a sample with known properties, make statistical inference to the larger population of venue visitors, and theorize about the introduction of biases that may limit generalization of results to the target population. The authors describe their use of TSS in the ongoing Community Intervention Trial for Youth (CITY) project to generate a systematic sample of young men who have sex with men. The project is an ongoing community level HIV prevention intervention trial funded by the Centers for Disease Control and Prevention. The TSS method is reproducible and can be adapted to hard-to-reach populations in other situations, environments, and cultures.

Recirculation of lymphocytes: its role in implementing immune responses in the skin.

A number of recent investigations have suggested that the subset of lymphocytes which migrate into nonlymphoid tissue to appear in peripheral lymph may be partly different from the major recirculating pool migrating through lymphoid tissues. We report results on the migrating from the blood of thoracic duct lymphocytes labelled with Cr and three subsets-accredited recirculators, activated lymphocytes and long-lived lymphocytes. The localization of these populations was studied in normal skin, in a contact sensitivity lesion and in a site of non-immune inflammation. All four populations localized in the contact sensitivity lesion in increased numbers compared to normal skin but long-lived lymphocytes appeared to discriminate between cell-mediated immunity and non-immune inflammation; activated lymphocytes migrated most efficiently into the non-immune inflammatory site (Table 4).