Treatment of metastatic melanoma with autologous, hapten-modified melanoma vaccine: regression of pulmonary metastases.

A human cancer vaccine composed of autologous tumor cells modified with the hapten dinitrofluorobenzene (DNP) induces cell-mediated immunity to the tumor cells and the development of inflammatory responses within metastatic sites. In this study we determined whether DNP vaccine could induce regression of established metastases. Ninety-seven patients (83 evaluable) with surgically incurable metastatic melanoma were treated with DNP vaccine preceded by low-dose cyclophosphamide. Tumor regression was assessed by standard criteria. The development of cell-mediated immunity to melanoma-associated antigens was measured by delayed-type hypersensitivity (DTH) testing before and after DNP vaccine treatment. Survival analysis was performed by the Kaplan-Meier method. There were 11 antitumor responses: 2 complete, 4 partial and 5 mixed. Both complete responses and 2 of the 4 partial responses occurred in patients with lung metastases. Response durations were as follows: partial responses-5, 6, 8 and 47+ months; and complete responses-12 and 29 months. Tumor regression required at least 4 months to become evident and in 2 cases maximum regression was not observed until 1 year after beginning treatment. Patients who exhibited tumor regression survived longer than those who did not (median survival times: responders, 21.4 months; non-responders, 8.7 months; p = 0.010). DTH to DNP-modified and unmodified autologous melanoma cells was induced in 87% and 42% of patients, respectively. The DTH response to unmodified cells was significantly associated with prolonged survival. Autologous DNP-modified melanoma vaccine can induce clinically meaningful regression of metastases and small lung metastases appear to be unusually sensitive. The development of DTH to unmodified, autologous tumor cells may be an important indicator of the vaccine's efficacy.

Antigen-specific humoral and cellular immune responses can be modulated in rhesus macaques through the use of IFN-gamma, IL-12, or IL-18 gene adjuvants.

DNA or nucleic acid immunization has been shown to induce both antigen-specific cellular and humoral immune responses in vivo. Moreover, immune responses induced by DNA immunization can be enhanced and modulated by the use of molecular adjuvants. To engineer the immune response in vivo towards more T-helper (Th)1-type cellular responses, we investigated the co-delivery of inteferon (IFN)-gamma, interleukin (IL)-12, and IL-18 genes along with DNA vaccine constructs. We observed that both antigen-specific humoral and cellular immune responses can be modulated through the use of cytokine adjuvants in mice. Most of this work has been performed in rodent models. There has been little confirmation of this technology in primates. We also evaluated the immunomodulatory effects of this approach in rhesus macaques, since non-human primates represent the most relevant animal models for human immunodeficiency virus (HIV) vaccine studies. As in the murine studies, we also observed that each Th1 cytokine adjuvant distinctively regulated the level of immune responses generated. Co-immunization of IFN-gamma and IL-18 in macaques enhanced the level of antigen-specific antibody responses. Similarly, co-delivery of IL-12 and IL-18 also enhanced the level of antigen-specific Th proliferative responses. These results extend this adjuvant strategy in a more relevant primate model and support the potential utility of these molecular adjuvants in DNA vaccine regimens.

Intratumoral recombinant GM-CSF-encoding virus as gene therapy in patients with cutaneous melanoma.

Seven immunocompetent, revaccinated patients with surgically incurable cutaneous melanoma underwent treatment of dermal and/or subcutaneous metastases with twice-weekly intratumoral injections of escalating doses (10(4)-2 x 10(7) plaque-forming units (PFU)/lesion; 10(4)-8 x 10(7) PFU/session) of a vaccinia/GM-CSF recombinant virus for 6 weeks. Patients with stable or responding disease were maintained on treatment until tumor resolution or progression. Systemic toxicity was infrequent, dose-related, and limited to mild flu-like symptoms that resolved within 24 hours. Local inflammation, at times with pustule formation, was consistently seen with doses of > or =10(7) PFU/lesion. Chronically treated lesions showed a dense infiltration, with CD4+ and CD8+ lymphocytes, histiocytes, and eosinophils. All seven patients developed an antivaccinia humoral immune response 14-21 days following revaccination. Despite the presence of these antivaccinia antibodies, the reporter gene was expressed, as judged by the development of anti-beta-galactosidase antibodies in all patients. Passenger cytokine gene function was evidenced by the presence of virally encoded GM-CSF mRNA at injection sites both early (weeks 1 and 5) and late (week 31) in the course of treatment. Eosinophilia at treatment sites indicated that physiologically significant levels of functional cytokine were generated. However, there were no changes in the total number of peripheral white blood cells or in the numbers or percentages of polymorphonuclear leukocytes, monocytes, or eosinophils. GM-CSF was not detected in the sera. The two patients with the largest tumor burdens failed to respond even at treatment sites. Three patients had mixed responses, with regression of treated and untreated dermal metastases and progression of disease elsewhere. One patient had a partial response, with regression of injected and uninjected regional dermal metastases. Residual melanoma was excised, rendering the patient disease free. One patient with only dermal metastases confined to the scalp achieved a complete remission. Sequential administration of escalating doses of a GM-CSF recombinant vaccinia virus is safe, effective at maintaining passenger gene function, and can induce tumor regression.

Enterovirus infections in England and Wales: laboratory surveillance data: 1975 to 1994.

Microbiology laboratories in England and Wales reported 40,366 culture confirmed isolates of echovirus (24,628; 61%) and coxsackievirus (B 11,714; 29%, A 4024; 10%) infections to the PHLS Communicable Disease Surveillance Centre (CDSC) in the 20 years from 1975 to 1994. Nearly half of the organisms were isolated from faeces, and 5741 were isolated from cerebrospinal fluid (75% of them echovirus, 13% coxsackie B, and 12% coxsackie A). Isolation rates for all enteroviruses were highest among infants aged 1 to 2 months. Sixty per cent of patients were aged under 5 years, 10% 5 to 9 years, and only 6% 35 years or over. Predominant serotypes were similar to those reported in other countries including the United States, Finland, and Belgium. Seventy-one per cent of reports were made between July and mid December. Periodicity varied between groups and serotypes: some demonstrated peaks at intervals of two to five years. There was evidence of spread of epidemic serotypes across Europe in certain years. Data collected between March and May each year enabled the strains circulating in the following 'season' to be predicted. Such information might be used to warn clinicians to anticipate particular clinical presentations.

Tumor-induced interleukin-10 inhibits type 1 immune responses directed at a tumor antigen as well as a non-tumor antigen present at the tumor site.

Interleukin (IL)-10 is a potent immunosuppressive cytokine that has been found to be present at the tumor site in a wide variety of human cancers, including transitional cell carcinoma of the bladder. Using a murine bladder tumor (MB49), which we show to express the male transplantation antigen (HY), we tested the hypothesis that IL-10 at the tumor site can block the generation of a tumor-specific type 1 immune response. We show that, despite its expression of HY, MB49 fails to prime for an HY-specific type 1 (IFN-gamma) response in normal female mice. Although MB49 does not constitutively produce IL-10, our data support a model whereby MB49 induces infiltrating cells to produce IL-10. This feature rendered the IL-10 knockout (KO) mouse, whose infiltrating cells are incapable of IL-10 production, a suitable model in which to study MB49 in the absence of IL-10. When injected into IL-10 KO mice, MB49 does prime for an HY-specific, type 1 immune response. Furthermore, IL-10 KO mice show prolonged survival and an increased capacity to reject tumors as compared with normal mice. We also tested the ability of tumor-induced IL-10 to inhibit immunization to a non-tumor antigen present at the tumor site. When vaccinia virus encoding beta-galactosidase (beta-gal) is injected into the tumors of normal mice, no beta-gal-specific IFN-gamma response is mounted. However, when this same viral construct is injected into the tumors of IL-10 KO mice, it produces a strong beta-gal-specific, IFN-gamma response. These studies demonstrate that tumor-induced IL-10 can block the generation of a tumor-specific type 1 immune response as well as subvert attempts to elicit a type 1 immune response to a non-tumor antigen at the tumor site.

Autologous, hapten-modified vaccine as a treatment for human cancers.

We have devised a novel approach to active immunotherapy based on modification of autologous cancer cells with the hapten, dinitrophenyl (DNP). The treatment program consists of multiple intradermal injections of DNP-modified autologous tumor cells mixed with BCG. Administration of DNP-vaccine to patients with metastatic melanoma induces a unique reaction- the development of inflammation in metastatic masses. Histologically, this consists of infiltration of T lymphocytes, most of which are CD8+. These T cells usually produce interferon-gamma in situ. Moreover, they represent expansion of T-cell clones with novel T-cell receptor (TCR) structures. Occasionally, administration of DNP-vaccine results in regression of measurable metastases. The most common site of regression has been small lung metastases. Administration of DNP-vaccine to patients in the postsurgical adjuvant setting produces a more striking clinical effect. Of 62 patients with clinically evident stage III melanoma who had undergone lymphadenectomy, the 5-year relapse-free survival rate was 45% and the overall survival rate was 58%. These results appear to be better than those obtained with high-dose interferon, although a randomized phase III trial is required to prove that point. A recent phase I study suggests that this therapeutic approach is also applicable to stage III ovarian cancer. There appear to be no insurmountable impediments to applying this approach to much larger numbers of patients or to developing it as a standard cancer treatment.

Shigella outbreak in a school associated with eating canteen food and person to person spread.

In June 1993 an outbreak of Shigella sonnei infection at a primary school in south east England affected 42% of 327 pupils and staff. Attack rates of diarrhoea and fever were 33% for children aged 4 to 8 years, and 8% for those aged 8 to 12 years (p < 0.00001). Illness was associated with eating canteen food (relative risk 5.9; 95% confidence interval 3.4, -10.3). All strains examined were S. sonnei phage type 3, with the same antibiogram (ttSTSS), and were indistinguishable using colicin typing and biotyping (colicin type 9, E8) and pulse field gel electrophoresis. Molecular epidemiology suggested but could not confirm that the outbreak strain was introduced into the school population from the community.

Coadministration of IL-12 or IL-10 expression cassettes drives immune responses toward a Th1 phenotype.

Cytokines are important regulators of the immune response. They influence immune expression, the development of immunologic memory, and regulation of antigen-specific and nonspecific immune activation as well as allergic responses. In a model system in mice, we have studied the effect of plasmids expressing interleukin (IL)-10 or IL-12 on the modulation of antigen-specific responses. Coadministration of IL-12 or IL-10 genes with DNA immunogens directed the antigen-specific immune response toward a T helper (Th1)-type immunity. In addition to the modulation of antigen-specific immune responses, we studied the induction of delayed-type hypersensitivity (DTH) to contact allergens as an in vivo model of the Th1 response. We found that IL-12 and IL-10 gene-containing plasmids, and not the bacterial plasmid alone, upregulate this response. Our cytokine gene delivery technique demonstrates an important level of control of the magnitude and direction of induced immune responses and could be advantageous in a wide variety of immunotherapeutic strategies.

STD and HIV screening in general practice: a survey related to termination of pregnancy in south Thames.

It has been recommended that screening for sexually transmitted disease (STDs) be carried out at the time of termination of pregnancy to allow prevention of post-abortion infection. Screening offers the opportunity to treat an infected woman and prevent serious complications following the termination procedure. Our findings indicate that general practitioners (GPs) in South Thames did not routinely carry out screening and few realized it took place at referral centres.

PIP: Recent research suggests the existence of a statistically significant association between sexually transmitted disease (STD) clinic attendance and previous pregnancy termination. Some have therefore recommended that women should be screened for infection with STDs at the same time they present to have their pregnancies terminated. In so doing, women infected with STDs can be identified and treated to prevent the potentially serious STD-related complications which may occur following pregnancy termination. A postal survey was sent to general practitioners (GPs) in South Thames to learn about the number of women they referred in 1994 for termination, their STD screening practice, knowledge of screening policy at referral centers, and follow-up arrangements post-termination. 62% of targeted GPs responded: 243 men, 113 women, and 12 practitioners of unknown sex. The GPs referred an estimated 2124 women for termination, of whom 71 (3%) were screened for STDs and 100 (5%) were reportedly tested for HIV (17 were positive). 4 GPs (1%) reported that they routinely screened, 18% that they sometimes screened, and 81% that they never screened. 27% of the female and 15% of the male GPs reported that they routinely or sometimes screened. Few GPs knew that such screening occurs at referral centers.

Phase I study of R24 in patients with metastatic melanoma including evaluation of immunologic parameters.

R24 is a mouse IgG3 monoclonal antibody with specificity for the disialoganglioside GD3. Most human melanomas have substantial surface GD3; in addition, a significant proportion of T lymphocytes display surface GD3. In a phase I study, we have investigated the toxicity and effect on selected immunological parameters of three dose levels of R24 given intravenously daily for five days (10 mg/m2/d, 30 mg/m2/d and 50 mg/m2/d) to patients with advanced melanoma. R24 administration neither consistently diminished nor augmented expression of delayed type hypersensitivity (DTH) skin reactions to anergy panel antigens or to a contact allergen dinitrofluorobenzene. R24 was infrequently found on tumor cells, or on lymphocytes from DTH biopsies, despite measurable serum levels of R24. The 30 mg/m2/d dose of R24 produced a statistically significant drop in peripheral blood lymphocytes on treatment Day 5. Likewise, on Day 5 there was a modest but statistically significant decrement in the proportion of circulating cells which were R24+. While there was one mixed response, there were no complete or partial tumor regressions in the R24 treated patients; there was no evident clinical benefit from the R24 therapy. The toxicity of the R24 at the higher dose levels can be very substantial. One patient, on the highest dose level, died on the 4th day of R24 treatment; in the absence of a plausible alternative explanation, a relationship of the death to the administered R24 must be considered. A precipitous drop in serum albumin coincident with R24 administration was found in all cases; this effect has not been previously reported with R24.

PHLS work on the surveillance and epidemiology of tuberculosis.

The re-emergence worldwide of tuberculosis as a major threat to public health and continuing and changing challenges in the control of tuberculosis in England and Wales provide the basis for the designation, by the PHLS, of tuberculosis as a priority area. In addition to the mycobacteriology reference services provided by the PHLS in England and Wales (summarised in an accompanying article) the PHLS contributes to the control of tuberculosis through its surveillance and other epidemiological work. This article summarises the range of this work, emphasising the collaborative nature of the effort required for surveillance, prevention, and control of tuberculosis.

Autologous hapten-modified melanoma vaccine as postsurgical adjuvant treatment after resection of nodal metastases.

PURPOSE: To determine whether treatment with an autologous whole-cell vaccine modified with the hapten dinitrophenyl (DNP vaccine) is an effective postsurgical adjuvant treatment for melanoma patients with clinically evident nodal metastases. PATIENTS AND METHODS: Eligible patients had regional nodal metastases that were large enough (> or = 3 cm diameter) to prepare vaccine. Following standard lymphadenectomy, patients were treated with DNP vaccine on a monthly or weekly schedule. RESULTS: Of 62 patients with metastasis in a single lymph node bed (stage III), 36 are alive after a median follow-up time of 55 months (range, 29 to 76); the projected 5-year relapse-free and overall survival rates are 45% and 58%, respectively. Of 15 patients with metastases in two nodal sites, five are alive with a median follow-up time of 73 months. An unexpected finding was the significantly better survival of older patients; the projected 5-year survival of patients greater than 50 versus < or = 50 years was 71% and 47%, respectively (P = .011, log-rank test). The development of a positive delayed-type hypersensitivity (DTH) response to unmodified autologous melanoma cells was associated with significantly longer 5-year survival (71% v 49%; P = .031). Finally, the median survival time from date of first recurrence was significantly longer for patients whose subcutaneous recurrence exhibited an inflammatory response (> 19.4 v 5.9 months; P < .001). CONCLUSION: Postsurgical adjuvant therapy with autologous DNP-modified vaccine appears to produce survival rates that are markedly higher than have been reported with surgery alone. Moreover, this approach has some intriguing immunobiologic features that might provide insights into the human tumor-host relationship.

Neutralizing anti-IL-10 antibody upregulates the induction and elicitation of contact hypersensitivity.

Various cytokines have been shown to modulate the acquisition and expression of delayed-type hypersensitivity. In a mouse model, we tested the notion that neutralization of interleukin-10 (IL-10), a cytokine that inhibits T cell-mediated reactions, would upregulate delayed-type hypersensitivity. We used two different monoclonal antibodies with specificity for murine IL-10 and used allergic contact dermatitis as a prototypical example of delayed-type hypersensitivity. When anti-IL-10 antibody was given at the time of sensitization to a contact allergen, there was a substantial increase in the induced contact hypersensitivity (CHS). In other experiments, the challenge reactions to contact allergen in routinely sensitized mice were increased when anti-IL-10 antibody was given at the time of challenge. Primary irritant reactions to croton oil were increased but only if anti-IL-10 antibody was given at the time of challenge and not when it was given a week previously. It appears that anti-IL-10 antibody can potentiate CHS reactivity by inactivating otherwise downregulating endogenous IL-10.

Active specific immunization in the treatment of patients with melanoma.

The bonafide albeit infrequent examples of tumor regression observed with whole tumor cell vaccines give evidence for the hypothesis that active specific immunization can induce a therapeutically effective immune response in melanoma patients. A dinitrophenyl-conjugated autologous whole tumor cell plus bacille Calmette-Guérin (BCG) vaccine administered in conjunction with low dose cyclophosphamide has produced clinically significant prolongation of disease free survival when used as a postsurgical adjuvant in patients with stage III melanoma. However, tumor cell-based vaccines are cumbersome and consequently of limited applicability. Improvements in our understanding of the antimelanoma immune response and technological advances have allowed investigators to explore better defined immunogens and antigenic targets; these include anti-idiotypic antibodies, gangliosides, and tumor associated/specific proteins and derived peptides. The rationale for, promise of, and progress to date with these materials are reviewed.

Cyclophosphamide and interleukin-12 synergistically upregulate the acquisition of allergic contact dermatitis in the mouse.

Cyclophosphamide given before allergen and recombinant interleukin-12 administered at the time of allergic sensitization substantially increase the acquisition of allergic contact dermatitis in the mouse. Since their immunoadjuvant mechanisms appeared different, it seemed probable that combining cyclophosphamide pretreatment with interleukin 12 administration would result in a more intense allergic contact dermatitis than when either agent was used alone. This was tested in different groups of mice sensitized to dinitrofluorobenzene or to oxazolone. Consistently, immunopotentiation of allergic contact dermatitis was significantly greater with the two immunoadjuvants than with either alone. This immunoadjuvant combination is likely to find use in immunization protocols designed to induce a Th-1 helper cell response.