Increased sexual activity reduces male immune function in Drosophila melanogaster

Despite the benefits of resistance, susceptibility to infectious disease is commonplace. Although specific susceptibility may be considered an inevitable consequence of the co-evolutionary arms race between parasite and host, a more general constraint may arise from the cost of an immune response. This “cost” hypothesis predicts a tradeoff between immune defense and other components of fitness. In particular, a tradeoff between immunity and sexually selected male behavior has been proposed. Here we provide experimental support for the direct phenotypic tradeoff between sexual activity and immunity by studying the antibacterial immune response in Drosophila melanogaster. Males exposed to more females showed a reduced ability to clear a bacterial infection, an effect that we experimentally link to changes in sexual activity. Our results suggest immunosuppression is an important cost of reproduction and that immune function and levels of disease susceptibility will be influenced by sexual selection.

Protein S is inducible by interleukin 4 in T cells and inhibits lymphoid cell procoagulant activity

Extravascular procoagulant activity often accompanies cell-mediated immune responses and systemic administration of pharmacologic anticoagulants prevents cell-mediated delayed-type hypersensitivity reactions. These observations suggest a direct association between coagulation and cell-mediated immunity. The cytokine interleukin (IL)-4 potently suppresses cell-mediated immune responses, but its mechanism of action remains to be determined. Herein we demonstrate that the physiologic anticoagulant protein S is IL-4-inducible in primary T cells. Although protein S was known to inhibit the classic factor Va-dependent prothrombinase assembled by endothelial cells and platelets, we found that protein S also inhibits the factor Va-independent prothrombinase assembled by lymphoid cells. Thus, protein S-mediated down-regulation of lymphoid cell procoagulant activity may be one mechanism by which IL-4 antagonizes cell-mediated immunity.

Midgut-specific immune molecules are produced by the blood-sucking insect Stomoxys calcitrans

We have cloned and sequenced two defensins, Smd1 and Smd2, from anterior midgut tissue of the blood-sucking fly Stomoxys calcitrans. The DNA and N-terminal protein sequences suggest both are produced as prepropeptides. Smd1 differs from the classic defensin pattern in having an unusual six-amino acid-long N-terminal sequence. Both Smd1 and Smd2 have lower pI points and charge than insect defensins derived from fat body/hemocytes. Northern analysis shows both of these defensin molecules are tissue specific; both are produced by the anterior midgut tissue and, unlike the other insect defensins reported to date, neither appears to be expressed in fat body or hemocytes. Northern analysis also shows that mRNAs for both defensins are constitutively produced in the anterior midgut tissues and that these transcripts are up-regulated in response to sterile as well as a lipopolysaccharide-containing blood meal. However, anti-Gram-negative biological activity in the midgut is substantially enhanced by lipopolysaccharide. These findings suggest that the insect midgut has its own tissue-specific immune mechanisms and that this invertebrate epithelium is, like several vertebrate epithelia, protected by specific antibacterial peptides.

Interferon-β gene therapy inhibits tumor formation and causes regression of established tumors in immune-deficient mice

Despite the potential of type 1 interferons (IFNs) for the treatment of cancer, clinical experience with IFN protein therapy of solid tumors has been disappointing. IFN-β has potent antiproliferative activity against most human tumor cells in vitro in addition to its known immunomodulatory activities. The antiproliferative effect, however, relies on IFN-β concentrations that cannot be achieved by parenteral protein administration because of rapid protein clearance and systemic toxicities. We demonstrate here that ex vivo IFN-β gene transduction by a replication-defective adenovirus in as few as 1% of implanted cells blocked tumor formation. Direct in vivo IFN-β gene delivery into established tumors generated high local concentrations of IFN-β, inhibited tumor growth, and in many cases caused complete tumor regression. Because the mice were immune-deficient, it is likely that the anti-tumor effect was primarily through direct inhibition of tumor cell proliferation and survival. Based on these studies, we argue that local IFN-β gene therapy with replication-defective adenoviral vectors might be an effective treatment for some solid tumors.

Tumor cells expressing a retroviral envelope escape immune rejection in vivo

A model system for the in vivo control of tumor cell proliferation by the immune system has been used to assay for the possible immunosuppressive activity of retroviral proteins. Expression vectors for the entire or the transmembrane subunit of the Moloney murine leukemia virus envelope protein were constructed, as well as control vectors for irrelevant transmembrane proteins—or no protein. They were introduced either into MCA205 murine tumor cells, which do not proliferate upon s.c. injection into an allogeneic host, or into CL8.1 murine tumor cells, which overexpress class I antigens and are rejected in a syngeneic host. In both cases, expression of the complete envelope protein or of the transmembrane subunit resulted in tumor growth in vivo, with no effect of control vectors. Tumor cell growth results from inhibition of the host immune response, as the envelope-dependent effect was no more observed for MCA205 cells in syngeneic mice or for CL8.1 cells in x-irradiated mice. This inhibition is local because it is not observed at the level of control tumor cells injected contralaterally. These results suggest a noncanonical function of retroviral envelopes in the “penetrance” of viral infections, as well as a possible involvement of the envelope proteins of endogenous retroviruses in tumoral processes.

The inhibition of antigen-presenting activity of dendritic cells resulting from UV irradiation of murine skin is restored by in vitro photorepair of cyclobutane pyrimidine dimers

Exposing skin to UVB (280–320 nm) radiation suppresses contact hypersensitivity by a mechanism that involves an alteration in the activity of cutaneous antigen-presenting cells (APC). UV-induced DNA damage appears to be an important molecular trigger for this effect. The specific target cells in the skin that sustain DNA damage relevant to the immunosuppressive effect have yet to be identified. We tested the hypothesis that UV-induced DNA damage in the cutaneous APC was responsible for their impaired ability to present antigen after in vivo UV irradiation. Cutaneous APC were collected from the draining lymph nodes of UVB-irradiated, hapten-sensitized mice and incubated in vitro with liposomes containing a photolyase (Photosomes; Applied Genetics, Freeport, NY), which, upon absorption of photoreactivating light, splits UV-induced cyclobutane pyrimidine dimers. Photosome treatment followed by photoreactivating light reduced the number of dimer-containing APC, restored the in vivo antigen-presenting activity of the draining lymph node cells, and blocked the induction of suppressor T cells. Neither Photosomes nor photoreactivating light alone, nor photoreactivating light given before Photosomes, restored APC activity, and Photosome treatment did not reverse the impairment of APC function when isopsoralen plus UVA (320–400 nm) radiation was used instead of UVB. These controls indicate that the restoration of APC function matched the requirements of Photosome-mediated DNA repair for dimers and post-treatment photoreactivating light. These results provide compelling evidence that it is UV-induced DNA damage in cutaneous APC that leads to reduced immune function.

Cyclin D3-associated Kinase Activity Is Regulated by p27kip1 in BALB/c 3T3 Cells

We report that cyclin D3/cdk4 kinase activity is regulated by p27kip1 in BALB/c 3T3 cells. The association of p27kip1 was found to result in inhibition of cyclin D3 activity as measured by immune complex kinase assays utilizing cyclin D3-specific antibodies. The ternary p27kip1/cyclin D3/cdk4 complexes do exhibit kinase activity when measured in immune complex kinase assays utilizing p27kip1-specific antibodies. The association of p27kip1 with cyclin D3 was highest in quiescent cells and declined upon mitogenic stimulation, concomitantly with declines in the total level of p27kip1 protein. The decline in this association could be elicited by PDGF treatment alone; this was not sufficient, however, for activation of cyclin D3 activity, which also required the presence of factors in platelet-poor plasma in the culturing medium. Unlike cyclin D3 activity, which was detected only in growing cells, p27kip1 kinase activity was present throughout the cell cycle. Since we found that the p27kip1 activity was dependent on cyclin D3 and cdk4, we compared the substrate specificity of the active ternary complex containing p27kip1 and the active cyclin D3 lacking p27kip1 by tryptic phosphopeptide mapping of GST-Rb phosphorylated in vitro and also by comparing the relative phosphorylation activity toward a panel of peptide substrates. We found that ternary p27kip1/cyclin D3/cdk4 complexes exhibited a different specificity than the active binary cyclin D3/cdk4 complexes, suggesting that p27kip1 has the capacity to both inhibit cyclin D/cdk4 activity as well as to modulate cyclin D3/cdk4 activity by altering its substrate preference.

Erythropoietin induces tumor regression and antitumor immune responses in murine myeloma models

Recombinant human erythropoietin (rHuEpo) has been used successfully in the treatment of cancer-related anemia. Clinical observations with several patients with multiple-myeloma treated with rHuEpo has shown, in addition to the improved quality of life, a longer survival than expected, considering the poor prognostic features of these patients. Based on these observations, we evaluated the potential biological effects of rHuEpo on the course of tumor progression by using murine myeloma models (MOPC-315-IgAλ2 and 5T33 MM-IgG2b). Here we report that daily treatment of MOPC-315 tumor-bearing mice with rHuEpo for several weeks induced complete tumor regression in 30–60% of mice. All regressors that were rechallenged with tumor cells rejected tumor growth, and this resistance was tumor specific. The Epo-triggered therapeutic effect was shown to be attributed to a T cell-mediated mechanism. Serum Ig analysis indicated a reduction in MOPC-315 λ light chain in regressor mice. Intradermal inoculation of 5T33 MM tumor cells followed by Epo treatment induced tumor regression in 60% of mice. The common clinical manifestation of myeloma bone disease in patients with multiple-myeloma was established in these myeloma models. Epo administration to these tumor-bearing mice markedly prolonged their survival and reduced mortality. Therefore, erythropoietin seems to act as an antitumor therapeutic agent in addition to its red blood cell-stimulating activity.

A mutant cholera toxin B subunit that binds GM1- ganglioside but lacks immunomodulatory or toxic activity

GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells. More recently, GM1 binding by isolated CtxB, or the related B subunit of Escherichia coli heat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes. Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action. We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57. Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity. The implications of these findings on the mode of action of cholera toxin are discussed.

Mechanism of activation for Zap-70 catalytic activity.

There is a growing body of evidence, including data from human genetic and T-cell receptor function studies, which implicate a zeta-associated protein of M(r) 70,000 (Zap-70) as a critical protein tyrosine kinase in T-cell activation and development. During T-cell activation, Zap-70 becomes associated via its src homology type 2 (SH2) domains with tyrosine-phosphorylated immune-receptor tyrosine activating motif (ITAM) sequences in the cytoplasmic zeta chain of the T-cell receptor. An intriguing conundrum is how Zap-70 is catalytically activated for downstream phosphorylation events. To address this question, we have used purified Zap-70, tyrosine phosphorylated glutathione S-transferase (GST)-Zeta, and GST-Zeta-1 cytoplasmic domains, and various forms of ITAM-containing peptides to see what effect binding of zeta had upon Zap-70 tyrosine kinase activity. The catalytic activity of Zap-70 with respect to autophosphorylation increased approximately 5-fold in the presence of 125 nM phosphorylated GST-Zeta or GST-Zeta-1 cytoplasmic domain. A 20-fold activity increase was observed for phosphorylation of an exogenous substrate. Both activity increases showed a GST-Zeta concentration dependence. The increase in activity was not produced with nonphosphorylated GST-Zeta, phosphorylated zeta, or phosphorylated ITAM-containing peptides. The increase in Zap-70 activity was SH2 mediated and was inhibited by phenylphosphate, Zap-70 SH2, and an antibody specific for Zap-70 SH2 domains. Since GST-Zeta and GST-Zeta-1 exist as dimers, the data suggest Zap-70 is activated upon binding a dimeric form of phosphorylated zeta and not by peptide fragments containing a single phosphorylated ITAM. Taken together, these data indicate that the catalytic activity of Zap-70 is most likely activated by a trans-phosphorylation mechanism.

Rational interleukin 2 therapy for HIV positive individuals: daily low doses enhance immune function without toxicity.

When administered in high doses to HIV positive (HIV+) individuals, interleukin 2 (IL-2) causes extreme toxicity and markedly increases plasma HIV levels. Integration of the information from the structure-activity relationships of the IL-2 receptor interaction, the cellular distribution of the different classes of IL-2 receptors, and the pharmacokinetics of IL-2 provides for the rationale that low IL-2 doses should circumvent toxicity. Therefore, to identify a nontoxic, but effective and safe IL-2 treatment regimen that does not stimulate viral replication, doses of IL-2 from 62,500 to 250,000 IU/m2/day were administered subcutaneously for 6 months to 16 HIV+ individuals with 200-500 CD4+ T cells/mm3. IL-2 was already detectable in the plasma of most HIV+ individuals even before therapy. Peak plasma IL-2 levels were near saturating for high affinity IL-2 receptors in 10 individuals who received the maximum nontoxic dose, which ranged from 187,500 to 250,000 IU/m2/day. During the 6 months of treatment at this dose range, plasma levels of proinflammatory cytokines remained undetectable, and plasma HIV RNA levels did not change significantly. However, delayed type hypersensitivity responses to common recall antigens were markedly augmented, and there were IL-2 dose-dependent increases in circulating Natural Killer cells, eosinophils, monocytes, and CD4+ T cells. Expanded clinical trials of low dose IL-2 are now warranted, especially in combination with effective antivirals to test for the prevention of immunodeficiency and the emergence of drug-resistant mutants and for the eradication of residual virions.

Structure-activity analysis of thanatin, a 21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides.

Immune challenge to the insect Podisus maculiventris induces synthesis of a 21-residue peptide with sequence homology to frog skin antimicrobial peptides of the brevinin family. The insect and frog peptides have in common a C-terminally located disulfide bridge delineating a cationic loop. The peptide is bactericidal and fungicidal, exhibiting the largest antimicrobial spectrum observed so far for an insect defense peptide. An all-D-enantiomer is nearly inactive against Gram-negative bacteria and some Gram-positive strains but is fully active against fungi and other Gram-positive bacteria, suggesting that more than one mechanism accounts for the antimicrobial activity of this peptide. Studies with truncated synthetic isoforms underline the role of the C-terminal loop and flanking residues for the activity of this molecule for which we propose the name thanatin.

Equine severe combined immunodeficiency: a defect in V(D)J recombination and DNA-dependent protein kinase activity.

V(D)J rearrangement is the molecular mechanism by which an almost infinite array of specific immune receptors are generated. Defects in this process result in profound immunodeficiency as is the case in the C.B-17 SCID mouse or in RAG-1 (recombination-activating gene 1) or RAG-2 deficient mice. It has recently become clear that the V(D)J recombinase most likely consists of both lymphoid-specific factors and ubiquitously expressed components of the DNA double-strand break repair pathway. The deficit in SCID mice is in a factor that is required for both of these pathways. In this report, we show that the factor defective in the autosomal recessive severe combined immunodeficiency of Arabian foals is required for (i) V(D)J recombination, (ii) resistance to ionizing radiation, and (iii) DNA-dependent protein kinase activity.