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Background There are limited data regarding immunological correlates of protection for the modified vaccinia Ankara (MVA) smallpox vaccine. Methods A total of 523 vaccinia-naive subjects were randomized to receive 2 vaccine doses, as lyophilized MVA given subcutaneously, liquid MVA given subcutaneously (liquid-SC group), or liquid MVA given intradermally (liquid-ID group) 28 days apart. For a subset of subjects, antibody-dependent cellular cytotoxicity (ADCC), interferon-γ release enzyme-linked immunospot (ELISPOT), and protein microarray antibody-binding assays were conducted. Protein microarray responses were assessed for correlations with plaque reduction neutralization titer (PRNT), enzyme-linked immunosorbent assay, ADCC, and ELISPOT results. Results MVA elicited significant microarray antibody responses to 15 of 224 antigens, mostly virion membrane proteins, at day 28 or 42, particularly WR113/D8L and WR101H3L. In the liquid-SC group, responses to 9 antigens, including WR113/D8L and WR101/H3L, correlated with PRNT results. Three were correlated in the liquid-ID group. No significant correlations were observed with ELISPOT responses. In the liquid-ID group, WR052/F13L, a membrane glycoprotein, correlated with ADCC responses. Conclusions MVA elicited antibodies to 15 vaccinia strain antigens representing virion membrane. Antibody responses to 2 proteins strongly increased and significantly correlated with increases in PRNT. Responses to these proteins are potential correlates of protection and may serve as immunogens for future vaccine development. Clinical Trials Registration NCT00914732.

The history, current status, opinions and attitudes, and ethical issues related to physician-assisted death (PAD) are examined. Specific implications for PAD and its interface with the human services professions are described with respect to multiculturalism; identity; perceptions of disease, illness, and pain; attitudes towards therapy; family decision making; ethics; and professional roles.

The authors provide reaction to the Major Contribution (MC) “Multicultural Clinical Supervision and Benchmarks: Empirical Support Informing Supervision Practice and Supervisor Training.” The article begins with an overview reaction to the MC. Following this, each of the four articles that compose the MC are discussed. The reactions provide both a contextual response to the articles as well a response to the central theme of the MC—that is, the intersection of supervision, multiculturalism, and the competency benchmarks.

Oligodeoxynucleotides containing CpG motifs (CpG ODN) can alter various immune cell subsets important in antibody therapy of malignancy. We undertook a phase I trial of CPG 7909 (also known as PF-3512676) in patients with previously treated lymphoma with the primary objective of evaluating safety across a range of doses, and secondary objectives of evaluating immunomodulatory effects and clinical effects. Twenty-three patients with previously treated non-Hodgkin lymphoma received up to 3 weekly 2-hour intravenous (IV) infusions of CPG ODN 7909 at dose levels 0.01 to 0.64 mg/kg. Evaluation of immunologic parameters and clinical endpoints occurred for 6 weeks. Infusion-related toxicity included grade 1 nausea, hypotension, and IV catheter discomfort. Serious adverse hematologic events observed more than once included anemia (2=Gr3, 2=Gr4), thrombocytopenia (4=Gr3), and neutropenia (2=Gr3), and were largely judged owing to progressive disease. Immunologic observations included: (1) The mean ratio of NK-cell concentrations compared with pretreatment at day 2 was 1.44 (95% CI=0.94-1.94) and at day 42 was 1.53 (95% CI=1.14-1.91); (2) NK activity generally increased in subjects; and (3) Antibody-dependent cellular cytotoxicity activity increased in select cohorts. No clinical responses were documented radiographically at day 42. Two subjects demonstrated late response. We conclude CpG 7909 can be safely given as a 2-hour IV infusion to patients with previously treated non-Hodgkin lymphoma at doses that have immunomodulatory effects.

A single ethanol ingestion of 1 g/kg by healthy individuals under controlled conditions does not inhibit and may stimulate fresh natural killer (NK) activity measured 16 hr later. However, ethanol inhibits fresh human NK activity when added to the lytic assay medium, as reported previously by other investigators. In contrast, using the same target (K562 erythroleukemia cells), peripheral blood mononuclear cells cultured 3 days with 50 units/ml of interleukin-2 are no longer inhibited significantly by the same concentration of ethanol that inhibited the fresh cells by 80%. When freshly isolated peripheral blood mononuclear cells, monocyte-depleted lymphocytes, or partially purified NK cells are pre-exposed to ethanol in vitro for 1 to 7 days, washed, and assayed for lytic activity against K562, the lytic activity is increased compared with nonethanol-exposed cells incubated concurrently. This increase is not dependent on accessory cells, added cytokines, or cell growth, and seems to be an intrinsic response of the NK subset to ethanol exposure. The finding of NK stimulation by ethanol, considered together with the observation of NK cell loss in some chronic alcoholics, suggests that loss of NK activity in the chronic alcoholic may result from cell loss rather than direct ethanol inhibition of NK activity.

Normal murine splenocytes cultured with IL2 for 6, but not 3, days contained an NK1.1 + , CD3 + lytically active subset. These lymphocytes were not derived from NK1.1 + precursors since NK1.1 + cells, purified by flow cytometry, failed to express CD3, as determined by the 145-2C11 mAb, on their surface even after culture with IL2 for 6 days. Instead, the precursors of the NKl.1 + , CD3 + effectors were contained in a B cell-depleted CD4 − , CD8 − , NK1.1 − splenic subset. Freshly obtained CD4 − , CD8 − , NK1.1 − splenocytes were mostly CD3 + , CD5 + , B220 − , had no spontaneous lytic activity against YAC-1, and were unable to mediate anti-CD3 directed lysis against FcR-bearing target cells. Culture of the CD4 − , CD8 − , NK1.1 − splenocytes with IL2, for 6 days, resulted in the development of NK1.1 + , CD3 + , B220 + effectors 40% of which were CD5 dim and 20–25% of which expressed TCR-β8 as determined by the F23.1 mAb. The acquisition of NK1.1, B220, and lytic activity by this triple-negative subset was readily inhibited by cyclosporine A (CSA). On the other hand, CSA had no effect on the acquisition of B220 or lytic activity by NK1.1 + precursors obtained by flow cytometry sorting. Moreover, all of the NK1.1 + cells generated by IL2 culture of splenocytes obtained from mice depleted of NK1.1 + lymphocytes (by in vivo injection of anti-NK1.1 mAb) coexpressed CD3 on their surface and were thus distinct from classical NK cells. These findings demonstrate tjiat splenic NK cells do not express or acquire CD3; that the NK1.1 + , CD3 + LAK effectors are derived from an NK1.1 − precursor; and that CSA is exquisitely selective in its inhibitory effect on LAK generation.

The Ly24 (Pgp-1) marker is expressed on some, but not all, mature T lymphocytes. It has recently become apparent that the development of Ly24− T lymphocytes is dependent on the presence of an intact thymus and that virgin Ly24− T cells rapidly acquire this marker upon antigenic or mitogenic stimulation. Although natural killer (NK) cells can develop and function in the absence of an intact thymus, some NK cell subsets express certain markers normally associated with T lymphocytes. The experiments in this report were undertaken to determine if NK cells express Ly24 and whether such an expression could be used to delineate distinct NK cell subsets. We found that mature functional NK cells expressed the Ly24 marker as defined by the monoclonal antibody 9F3. Double-color fluorescence analysis using C57BL/6 splenocytes (whose NK cells express the NK1.1 marker) showed all the NK1.1+ cells to be Ly24+ as well. For C3H/HeN (an NK 1.1− strain), double-color fluorescence analysis utilizing asialo GM1 and Ly24 revealed a distinct subset positive for both markers and containing most of the functional NK cell activity. Whereas the Ly24 marker did not illuminate an NK cell subset, these findings demonstrate that this determinant can be useful for the further characterization and isolation of NK cells.

Oligodeoxynucleotides (ODN) with the CpG motif have been shown to be potent stimulators of innate immunity. A theoretical concern is that uncontrolled stimulation of the innate immune system through the TLR-9 receptor could induce, or worsen, some autoimmune diseases such as adjuvant arthritis or systemic lupus erythematosus. Safe therapeutic use of such ODN could be enhanced if one could regulate some of their stimulatory activities. We have designed a group of synthetic ODNs, which were able to inhibit the induction of NK lytic activity, IL-12p40 and IFN-gamma cytokine secretion by type A (D)-CpG-ODNs. Inhibition occurred in both DNA-sequence and dose-dependent fashion. Fifty percent inhibition was achieved with ~10-nM concentration of the most potent inhibitory ODNs. Delayed addition of these ODNs for up to 2 h was still able to profoundly affect CpG-induced IL-12p40 production at 18 h. Inhibitory DNA motif consists of two nucleotide triplets, a proximal pyrimidine-rich CCT sequence and a more distal GGG triplet. Optimal distance between these blocks is between three to five nucleotides. The linker sequence between the CCT and GGG blocks can additionally modify the activity of inhibitory ODNs, in both a positive and in negative way. When the order of CCT and GGG blocks is reversed, inhibition is completely lost. These findings suggest that CpG regulation of innate immunity can itself be regulated by particular motifs, which could be of therapeutic benefit in autoimmune diseases.