Natural cytotoxicity of NC-2+ cells against the growth and metastasis of WEHI-164 fibrosarcoma.

We have previously reported a new receptor (NC-2) for natural cytotoxicity (NC) on murine leucocytes, identified by monoclonal antibody D9 (mAb D9). Pretreatment of mouse spleen cells with different concentrations of mAb D9 in vitro blocked NC against WEHI-164, whereas natural killing (NK) activity against YAC-1 was unaffected. This paper reports the immune surveillance against the growth of WEHI-164 tumour cells in mice by NC-2(+) Cells. The kinetics of in vivo reduction in NC activity were investigated by treating BALB/c and (CBA × C57BL/6) F1 mice with a single injection of 40 µg of mAb D9 and monitoring splenic NC activity by (51) Cr-release assay at intervals from 24 h to 3 weeks. Control mice were injected with OKT8 irrelevant antibody. Results showed a significant (P < 0.05) reduction in splenic NC activity within 24 h which persisted for up to 1 week. Similar results were also obtained when (CBA × C57BL/6) F1 mice were employed (P<0.001). In vivo tumour studies were undertaken to investigate the role of NC-2(+) cells in surveillance against tumour growth and metastasis of the WEHI-164 fibrosarcoma. When syngeneic BALB/c mice were injected with 40 µg of mAb D9 and then challenged with 5 × 10(5) WEHI-164 cells, results showed significantly increased growth rate of the transplanted WEHI-164 fibrosarcoma and tumour nodules in the lungs of animals, when compared to control mice with normal NC activity. Our data support an innate surveillance in metastasis and growth of WEHI-164 fibrosarcoma in mice.

Double-Dose Desogestrel: Is it Effective in Adolescent Menstrual Dysfunction?

STUDY OBJECTIVE: Adolescent menstrual dysfunction (AMD) is a common cause of iron deficiency anemia and absences from school. The management of AMD with single- and double-dose desogestrel is largely on the basis of anecdotal evidence. Our aim was to describe the effectiveness and safety of both dosing strategies in our clinic cohort to help guide future management. DESIGN: Local service evaluation with retrospective analysis of clinic notes. SETTING: Adolescent gynecology clinic in a tertiary pediatric center in the North West of England. PARTICIPANTS: Adolescent girls (10-18 years of age) with AMD (n = 129). INTERVENTIONS: Single-dose (75 µg) desogestrel vs double-dose (150 µg) desogestrel. MAIN OUTCOME MEASURES: Prevalence of amenorrhea and light spotting, side effects, and discontinuation rates of both dosing regimens. RESULTS: Forty-three of 87 (49%) adolescent girls who started treatment with a double dose of desogestrel were amenorrheic/experienced light spotting, compared with 7/40 (18%) of girls who started treatment with a single dose (P = .001). Patients taking a double dose of desogestrel were less likely to discontinue overall (double: 45/89 [51%]; vs single: 35/40 [88%]; P < .001) and there was no evidence of an increase in nonbleeding side effects (double: 30/89 [34%]; vs single: 15/40 [38%]; P = .68). CONCLUSION: Our findings provide evidence that a double dose of desogestrel is associated with a higher prevalence of amenorrhea and light spotting compared with a single dose in adolescent girls with AMD. However, larger studies are needed to further inform clinical guidelines.

Heterogeneity of natural killer cells in the mouse.

Mice were treated with the bone-seeking isotope, 89Sr, cyclophosphamide, and short-term lethal irradiation in vivo, and murine spleen cells are treated with anti-Nk-1.2 plus complement (C) in vitro. Fresh spleen cell suspensions from the above groups and from beige and neonatal mice were subsequently tested for natural killer (NK) cell activity against a panel of lymphoid and nonlymphoid tumor cell target. NK cell reactivities against YAC-1, MPC-11, and Cl.18 tumors were markedly and consistently reduced in (a) mice treated with 89Sr, (b) spleen cells treated with anti-Nk-1.2 plus C, and (c) C57BL/6 bg/bg mice. In contrast, NK activities against FLD-3 and WEHI-164.1 tumors were usually normal in mice treated with 89Sr, in beige mutant mice, and in spleen cells after treatment with anti-Nk-1.2 antibody and C. It appears, therefore, that two major groups of NK cells exist in fresh mouse spleen cells suspensions. NK-A cells are marrow dependent, Nk antigen positive, and deficient in beige mice; these lyse YAC-1, MPC-11, and Cl.18 tumors. NK-B cells, which are responsible for the lysis of WEHI-164.1 and FLD-3, are Nk antigen negative, marrow independent, and unaffected by the bg/bg mutation. Other features of NK-B cells, suggest that these NK cells, although they share the characteristics mentioned above, differ among themselves especially with respect to age of maturation and susceptibility to cyclophosphamide and total body irradiation. The NK-B group may therefore induce subsets that remain to be defined.

Mechanisms of syngeneic tumor rejection. Susceptibility of host-selected progressor variants to various immunological effector cells.

The ultraviolet radiation-induced fibrosarcoma 1591 generally is rejected by normal syngeneic mice and only rarely exhibits progressive growth. We isolated five of these rare progressor tumors from normal animals to determine the selective pressures that had been exerted upon the parental tumor by normal immunocompetent hosts. We found that the variant tumor cell lines could neither induce nor be killed by tumor-specific lymphocytes, suggesting that selection had been exerted against tumor cells expressing the tumor-specific antigen. In contrast, no selection against natural killer cell activity or against nonspecific T cell-mediated immunity seems to have occurred because progressor tumor cells were highly sensitive to these types of effector cells and in fact induced these effector cells more effectively than did the parental tumor. Nude mice were found to be as capable as normal mice in generating natural killer activity in response to a challenge with progressor tumor cells, but they were unable to mount a nonspecific T lymphocyte response. This may account for the fact that the progressor tumors grew at a significantly faster rate in nude animals than in normal mice. Thus, our study shows that in this tumor system nonspecific T cell-mediated immunity may play a role in retarding tumor growth, but the absolute resistance of normal animals to progressive tumor growth critically depends upon the presence of T cell-mediated tumor-specific immunity. Furthermore, neither NK cells nor nonspecific cytotoxic T lymphocytes appear to play a role in immunoselection against this tumor in normal immunocompetent hosts.

Impact of dermoscopy and short-term sequential digital dermoscopy imaging for the management of pigmented lesions in primary care: a sequential intervention trial.

BACKGROUND: Studies have shown the benign to malignant ratio of excised pigmented skin lesions is suboptimal in primary care. OBJECTIVES: To assess the impact of dermoscopy and short-term sequential digital dermoscopy imaging (SDDI) on the management of suspicious pigmented skin lesions by primary care physicians. METHODS: A total of 63 primary care physicians were trained in the use of dermoscopy and SDDI (interventions) and then recruited pigmented lesions requiring biopsy or referral in routine care by naked eye examination. They were then given a dermatoscope and the option of a SDDI instrument, and change of diagnosis and management was assessed. RESULTS: Following the use of the interventions on 374 lesions a total of 163 lesions (43.6%) were excised or referred, representing a reduction of 56.4%. Of the 323 lesions confirmed to be benign, 118 (36.5%) were excised or referred, leading to a reduction of 63.5% (P < 0.0005) in those requiring excision or referral. The baseline naked eye examination benign to melanoma ratio was 9.5 : 1 which decreased to 3.5 : 1 after the diagnostic interventions (P < 0.0005). Of the 42 malignant lesions included in the study (34 melanoma, six pigmented basal cell carcinoma and two Bowen disease) only one in situ melanoma was incorrectly managed (patient to return if changes occur) resulting in the correct management of 97.6% and 97.1% of malignant pigmented lesions and melanoma, respectively. CONCLUSIONS: In a primary care setting the combination of dermoscopy and short-term SDDI reduces the excision or referral of benign pigmented lesions by more than half while nearly doubling the sensitivity for the diagnosis of melanoma.

Relationship between solution structure and phase behavior: a neutron scattering study of concentrated aqueous hexamethylenetetramine solutions.

The water-hexamethylenetetramine system displays features of significant interest in the context of phase equilibria in molecular materials. First, it is possible to crystallize two solid phases depending on temperature, both hexahydrate and anhydrous forms. Second, saturated aqueous solutions in equilibrium with these forms exhibit a negative dependence of solubility (retrograde) on temperature. In this contribution, neutron scattering experiments (with isotopic substitution) of concentrated aqueous hexamethylenetetramine solutions combined with empirical potential structure refinement (EPSR) were used to investigate the time-averaged atomistic details of this system. Through the derivation of radial distribution functions, quantitative details emerge of the solution coordination, its relationship to the nature of the solid phases, and of the underlying cause of the solubility behavior of this molecule.

Tumor immunity to murine plasma cell tumors. V. Genetic control of the in vitro cytotoxic T-cell response to plasma cell tumor-associated antigens of NZB mice.

Cytotoxic T-lymphocytes (Tc) were induced in vitro to plasmacytoma tumor-associated antigens (TAA) by coculture of irradiated cells of plasma cell tumors (PCT) from NZB mice and viable nonimmune or PCT-immune spleen cells from NZB. (NZB X C57BL)F1, (NZB X B10.D2)F1, and (NZB X B10,BR)F1 mice. When nonimmune spleen cells were used, major histocompatibility complex (MHC)-linked genetic control of te primary in vitro induction of Tc to NZB PCT was demostrated for a shared TAA expressed on NZB and BALB/c PCT. Evidence was also obtained for a non-MHC-linked genetic control of the primary in vitro induction of Tc to a second TAA that ws expressed on both PCT and T-lymphomas. When the spleen cells were obtained from mice preimmunized to an NZB PCT, a secondary in vitro Tc response was observed, and a PCT-specific and strain-specific TAA or NZB mice was identified. In addition, results with the F1 hybrids also indicated an MHC-linked genetic control of the in vitro Tc response to this strain-specific TAA.

Tumor immunity to murine plasma cell tumors. III. Detection of common and unique tumor-associated antigens on BALB/c, C3H, and NZB plasmacytomas by in vivo and in vitro induction of tumor-immune responses.

Tumor-associated antigens (TAA) were demonstrated on plasmacytomas derived from BALB/c, NZB, and C3H mouse strains, by in vivo and in vitro techniques. By immunizing the appropriate F1 hybrid mice with these tumors, it was possible to show that all the plasmacytomas expressed cross-reactive tumor-associated transplantation antigens. When cytotoxic lymphocytes (CL) were induced in vitro by the coculturing of syngeneic or F1 hybrid spleen cells with irradiated plasmacytoma cells, "shared" and "unique" plasmacytoma TAA were demonstrable. This was accomplished by inducing CL in vitro against one syngeneic plasmacytoma and assaying for lytic activity on a range of 51Cr-labeled BALB/c, NZB and C3H plasmacytoma cells in vitro. In a second in vitro assay, unlabeled plasmacytoma cells were tested for their ability to inhibit the lysis of a particular 51Cr-labeled plasmacytoma, with the use of CL induced in vitro against it. The possibility that these TAA were "self" antigens was excluded by demonstrating in the inhibition assay that they were not present on T lymphomas and spleen cells of the same strain, and that CL "autosensitized" in vitro could not significantly lyse 51Cr-labeled plasmacytoma cells in vitro. From both in vivo and in vitro studies of immunity to these tumors, it was concluded that any one plasmacytoma line possesses multiple TAA of both shared and unique types.

In vitro induction of tumor-specific immunity. II. Activation of cytotoxic lymphocytes to murine oncofetal antigens.

The presence of oncofetal antigens (OFA) on a wide variety of murine tumor cells was demonstrated to a totally in vitro system of cellular immunity. Nonimmune spleen lymphocytes were cocultivated with irradiated syngeneic fetal liver cells and, at various times after initiation of culture, were tested for the presence of cytotoxic lymphocytes (CL) by 51Cr-release assay with labeled tumor target cells. Significant cytotoxic activity was regularly detected after such culture, whereas only minor levels appeared in control cultures of spleen lymphocytes with irradiated syngeneic spleen cells. Specificity of the reaction was assessed by inhibition tests in which nonlabeled cells were admixed to the CL and 51Cr-labeled tumor targets. Fetal liver cells gave significant inhibition; however, no inhibition was found with adult spleen cells. Various tumor types gave inhibition, and fibrosarcomas were more effective than plasmacytomas or lymphomas. The results suggested that all tumor types tested possess such OFA, as well as their unique or virus-associated, tumor-associated transplantation antigens, and that the in vitro system permits a more active response to the tumor-associated OFA than that observed in in vivo studies.

In vitro induction of tumor-specific immunity. VI: analysis of specificity of immune response by cellular competitive inhibition: limitations and advantages of the technique.

The cellular competitive inhibition 51Cr-release assay makes two distinct contributions to the in vitro study of cell-mediated immunity. It allows target cells which are not amenable to isotopic labelling to be investigated for their antigenic specificity, and it provides a means, complementary to the direct cytotoxicity assay, of estimating qualitative and quantitative differences in antigen expression on intact normal and neoplastic cells. Various parameters of a micro-51Cr-release inhibition assay have been studied, and it was found that the assay conditions markedly influenced both the sensitivity and specificity. It is concluded that optimal assay conditions for specificity include: 1) moderate levels of lysis on the linear part of the CL/T titration curve, 2) avoidance of prolonged assay times, and 3) low ratios of blocker to target cells. When tumor cells with large cell volumes are used as competitive inhibitor (blocker) cells, non-specific blocking will occur; limits have been defined for this particular micro-inhibition assay which, in general, exclude these effects.

T cell responses to alloantigens. III. Prolonged circulation of specific cytotoxic T memory cells following skin allograft rejection in mice.

A limit dilution technique was used to study specific circulating cellular memory to alloantigens in the blood of B6AF1 mice that had rejected B10.D2 or B10.BR skin grafts. A specific increase in cytotoxic T cell (Tc) precursor (CTL-P) frequency was detected as the skin grafts were rejected, and this persisted for the duration of the experiment (8 months) in the B6AF1 mice that had rejected B10.D2 skin grafts. By contrast, the CTL-P frequency for H-2Dk in the blood of B6AF1 mice that had rejected B10.BR skin grafts had returned to normal by 4 months after graft rejection. In addition, it was shown that peripheral blood lymphocytes from these immune mice could respond to heat-inactivated spleen cells of donor origin, whereas those of naive mice could not.

T cell responses to alloantigens. II. In vitro induction of specific Ts by heat-inactivated allogeneic lymphoid cells.

When viable B6AF1 murine spleen cells were cultured in combination with heat-inactivated B10.D2 spleen cells under appropriate conditions, alloantigen-specific suppressor T cells (Ts) were generated. The optimal conditions for generation of specific Ts included a responder cell number of 2 to 4 X 10(6)/ml, a high stimulator to responder ratio, the use of at least 30 min of heat treatment (45 C) of stimulator cells, and 6 or more days of culture.

Maternal alloimmunisation in pregnancy. In vitro studies of T cell-dependent immunity to paternal alloantigens.

A secondary in vitro allograft reaction was used to demonstrate that spleen cells derived from allogeneically mated inbred mice were immunised against paternal alloantigens. In addition to the heightened alloantigen-specific in vitro response of these spleen cells, it was also found that spleen cells froma a wide variety of syngeneically and allogeneically mated mice were nonspecifically more reactive in the in vitro allograft reaction than spleen cells from virgin mice. However, when spleen cells freshly harvested from allogeneically mated mice were tested in a direct 51Cr release assay, lysis of target cells bearing the paternal alloantigens was demonstrable in only one-third of the experiments. It is proposed that T cell immunisation to paternal alloantigens occurs in pregnancy, but that cell-mediated cytotoxicity is inhibited.

T cell responses to alloantigens. IV. In vivo and in vitro studies of the abrogation of presensitization to major histocompatibility antigens.

B6AF1 mice were sensitized to major histocompatibility complex (MHC) antigens by rejection of B10.D2 skin allografts. The effects of various protocols of treatment with antithymocyte serum (ATS), Bordetella pertussis vaccine, and donor strain lymphocytes were tested in these mice, and the results were assessed by in vitro testing for abrogation of circulating cytotoxic T cell memory and in vivo by determining the survival time of a second donor strain skin graft. It was found that a 6-week course of ATS alone abrogated this presensitized state as judged by both the in vitro and in vivo measurements, and that the hyporesponsive state induced by this treatment persisted for at least 4 months.

Treatment of acute renal allograft rejection with OKT3 monoclonal antibody.

Eight cadaver donor renal allograft recipients, who had received azathioprine and prednisone from the day of transplantation, were treated with OKT3 monoclonal antibody (reactive with all mature peripheral blood T cells) at the time of diagnosis of acute rejection. In all cases, loss of essentially all detectable peripheral blood OKT3-reactive cells was noted within minutes after the initial 1- to 5-mg i.v. infusion. Chills and fever invariably occurred following the first or second infusion of monoclonal antibody, but were not noted during the subsequent, 10- to 20-day course of therapy, suggesting rapid cell lysis as the etiology of this toxicity. The established rejection episode was reversed in all cases within 2 to 7 days without addition of any therapy other than OKT3 antibody and despite continued lowering of the steroid dosages. During the subsequent 3- to 12-month follow-up period, further rejection episodes occurred in five of these patients, two of these were irreversible with conventional therapy so that six of the eight allografts continue with excellent renal function. These preliminary observations suggest that homogeneity, limited dosage requirements, and ease of in vitro monitoring of dosage effects should markedly simplify the use of monoclonal antibody to T cell populations in human allograft recipients. This second generation of antilymphocyte preparations offers the potential for not only increased effectiveness but also the possibility of manipulating specific T cell subsets.

Recent incidence trends imply a nonmetastasizing form of invasive melanoma.

In the mid- to late-1980s white populations in Australia, New Zealand and Scotland showed a sharp increase in melanoma incidence above preceding long-term trends, in some cases as much as doubling in as little as 2 years. Most of this increase was in thin melanomas, (< 1.50 mm thick), and males were more affected than females. Thicker melanomas also generally increased in incidence, particularly in males aged 65 years or older. Examination of Australian Medicare and pathology laboratory data indicated that excision of skin lesions and laboratory diagnosis of pigmented lesions also rose sharply in this period, suggesting that advancement of the time of diagnosis was a likely factor in the increase in melanoma incidence. However the maintenance of new higher incidence levels and the increase in incidence of thicker lesions suggests that advancement of diagnosis cannot explain all of the increase. A real increase in incidence and increasing diagnosis of a preexisting, non-metastasizing form of thin melanoma may also have contributed.

Effects of prophylactic antibiotics on wound infection after elective colon and rectal surgery: 1960 to 1980.

Wound infection continues to be a common complication of elective colon and rectal surgery. During the period from 1960 to 1980, 42 prospective, controlled prophylactic antibiotic trials were undertaken which addressed this problem. In this report we have analyzed these trials and compared them to all noncontrolled, prospective wound infection surveys and a representative sample of the retrospective surveys of the same period. From this analysis several conclusions have become apparent: (1) wound infection remains a common complication for which prophylactic antibiotics are generally effective, (2) the most effective agents are those with activity against anaerobic bacteria, (3) orally administered nonabsorbable antibiotics have little effect on reducing wound infection following these procedures, and (4) the optimal antibiotic regimen is yet to be found. The data do suggest, however, the more preferred regimens currently available as well as those worthy of further investigation.

Screening for melanoma by primary health care physicians: a cost-effectiveness analysis.

BACKGROUND AND DESIGN - Australia has the highest rates of skin cancer in the world, and the incidence is estimated to be doubling every 10 years. Despite advances in the early detection and treatment of melanoma about 800 people still die nationally of the disease each year. A possible strategy for further reducing the mortality from melanoma is an organised programme of population screening for unsuspected lesions in asymptomatic people. Arguments against introducing melanoma screening have been based on cost and the lack of reliable data on the efficacy of any screening tests. To date, however, there has been no systematic economic assessment of the cost effectiveness of melanoma screening. The purpose of this research was to determine whether screening may be potentially cost effective and, therefore, warrants further investigation. A computer was used to simulate the effects of a hypothetical melanoma screening programme that was in operation for 20 years, using cohorts of Australians aged 50 at the start of the programme. Based on this simulation, cost-effectiveness estimates of melanoma screening were calculated. RESULTS - Under the standard assumptions used in the model, and setting the sensitivity of the screening test (visual inspection of the skin) at 60%, cost effectiveness ranged from Aust$6853 per life year saved for men if screening was undertaken five yearly to $12 137 if screening was two yearly. For women, it ranged from $11 102 for five yearly screening to $20 877 for two yearly screening. CONCLUSION - The analysis suggests that a melanoma screening programme could be cost effective, particularly if five yearly screening is implemented by family practitioners for men over the age of 50.