Relevance of CD38 expression on CD8 T cells to evaluate antiretroviral therapy response in HIV-1-infected youths.

Surrogate markers for monitoring immuno-virological discordant responders, in addition to plasma viral load and CD4 cells, are still lacking. We assessed the diagnostic utility of CD38 expression on CD8 T cell assay, alone or in association with lymphocyte proliferation to mycotic antigens, in evaluating antiretroviral response. 28 vertically HIV-infected youths, 21 HAART- and seven 2 nucleotide reverse transcriptase inhibitors-treated, were enrolled in a retrospective study. Responders (57.1%) and non-responders (42.9%) to stable antiretroviral therapy for a minimum of 6 months, on the basis of viral load and CD4 T cells, comprehensively evaluated by CD38 expression on CD8 T lymphocytes [measured as CD38 antibody bound per CD8 T cell (CD38 ABC) and %CD38+ of total CD8 T cells (%CD38/CD8)] and lymphocyte proliferation to P. jiroveci, C. albicans, C. neoformans, A. fumigatus at a single time point after treatment, were selected. CD38 expression > or =2401 CD38 ABC and > or =85% CD38/CD8 cut-off points, accurately discriminates responders versus non-responders, both measures resulting in 75.0% (CI 42.8-94.5) sensitivity (identification of non-responder) and 93.8% (CI 69.8-99.8) specificity (identification of responder), when considered as single assays. The association '> or =2401 CD38 ABC or > or =85% CD38/CD8' improved sensitivity to 83.3% (CI 51.6-97.9), while the association '<2401 CD38ABC (or <85% CD38/CD8) and lymphoproliferative response positive to > or =2 tested organisms' improved specificity to 100% (CI 79.4-100). In conclusions, CD38 expression and mycotic antigen-specific T-cell proliferation may be used as additional parameters to existing criteria to evaluate antiretroviral response in immuno-virological discordant patients.

Nitazoxanide or CD3+/CD4+ lymphocytes for recovery from severe Cryptosporidium infection after allogeneic bone marrow transplant?

We describe a case of Cryptosporidium infection occurring in a child after allogeneic SCT for acute non-lymphoblastic leukemia. This patient presented an intestinal, biliar, and pancreatic Cryptosporidium disease associated with an intestinal aGvHD. The increase in CD3+/CD4+ cells secondary to the reduction of steroid therapy associated with the improvement of aGvHD and the use of antiparasitic treatments (especially nitazoxanide) improved the infection-related symptoms and led to a complete clearance of the Cryptosporidium.

Antagonistic activity of HIV-1 T helper peptides flanked by an unrelated carrier protein.

Antagonism is the ability of a modified antigenic peptide (altered peptide ligand, APL) to prevent CD4 T cell activation by the original peptide. Here we show that antagonistic activity can be conferred to peptides of HIV envelope glycoprotein gp120 and reverse transcriptase p66 by adding flanking polypeptide sequences at the C or at the N terminus by genetic engineering, rather than by introducing substitutions by synthesis. The glutathione S-transferase (GST)-peptide system has been used to produce molecules that display the peptide at the appropriate end of the GST carrier. When the gp120 peptide 191-205 (pep24) was expressed at the C terminus of GST (GST-24), antigenicity of specific human CD4 T cells was maintained. In contrast, when the peptide was expressed at the N terminus of GST (24-GST), antigenicity was abolished and antagonistic activity was introduced. Similar results were obtained with a p66-derived peptide at the C terminus of the GST carrier. Antagonism was (1) specific; proliferation of a CD4 T cell line from the same donor responding to the envelope glycoprotein of another retrovirus, HTLV-1, was not affected; (2) reversible; proliferative response was rescued in T cells exposed to antigen-presenting cells (APC) pulsed with the antagonist; (3) dominant; T cells cultured with APC pulsed with the agonist and with APC pulsed with the antagonist did not proliferate. The carrier could be cleaved by proteolysis while the antagonistic activity was preserved. Thus a minimal sequence that confers antagonistic activity can be engineered or synthesized with peptides to antagonize undesired CD4 responses as an alternative to the use of APL.

Recognition of antigenic clusters of Candida albicans by T lymphocytes from human immunodeficiency virus-infected persons.

The fine specificity of the cellular immune response to Candida albicans (i.e., recognition of different antigenic components) between normal controls and human immunodeficiency virus-infected patients in various stages of disease was compared. C. albicans-specific T cells, enriched by antigen stimulation and interleukin-2 expansion, were challenged with antigenic fractions of different molecular weight obtained by SDS-gel fractionation of C. albicans extracts in the presence of autologous mononuclear cells as antigen-presenting cells. Proliferative responses showed similar patterns of reactivity between controls and category A and B seropositive subjects. Category C patients with concurrent C. albicans infections did not give rise to C. albicans-specific T cell lines, confirming the T cell defect. Patients without clinically evident C. albicans infection had a low but broad reactivity pattern of C. albicans-specific T cells. These results suggest that depletion of C. albicans-specific T cells, independent of their fine specificity, occurs along with disease progression.

Lack of alachlor induced DNA damage as assayed in rodent liver by the alkaline elution test.

Alachlor was studied in vivo for its capability to induce DNA damage, as evaluated by the alkaline elution test. The experiments were performed in mouse and rat liver after acute or subacute intraperitoneal or per os administrations of the chemical at sublethal dosages. Rat liver was also studied for DNA damage after administration of 2,6-diethylaniline, one of alachlor's major metabolites. Eluted DNA from treated animals was indistinguishable from control DNA. The results show that neither alachlor nor its metabolite cause DNA damage as determined by the number of single strand breaks.

Repair of DNA methylphosphotriesters through a metalloactivated cysteine nucleophile.

The Escherichia coli Ada protein repairs methylphosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteines. Upon methyl transfer, Ada acquires the ability to bind specific DNA sequences and thereby to induce genes that confer resistance to methylating agents. The amino-terminal domain of Ada, which comprises the methylphosphotriester repair and sequence-specific DNA binding elements, contains a tightly bound zinc ion. Analysis of the zinc binding site by cadmium-113 nuclear magnetic resonance and site-directed mutagenesis revealed that zinc participates in the autocatalytic activation of the active site cysteine and may also function as a conformational switch.

A genotoxicity study on N-acryloyl-N'-phenylpiperazine: implications of aneugenic effects in risk evaluations.

Further to a previous genotoxicity study, we analyzed sister-chromatid exchange (SCE) and DNA-repair induction (V79 and EUE cells in vitro) and DNA damage (rat liver in vivo) with regard to N-acryloyl-N'-phenylpiperazine (AcrNPP), a chemical proposed for biomaterial polymerization which contains an aromatic tertiary amine function in a piperazine cycle. This chemical induced SCEs in a dose-dependent fashion (up to approximately 3.7 times the control value), while it was negative for DNA-repair induction and weakly yet significantly positive for in vivo DNA damage (maximum increase approximately 1.4 times the control value). Taken together with our previous genotoxicity data on AcrNPP and structurally related compounds, the present results confirm that aneuploidy is a possible major effect of aromatic tertiary amines. As regards exposure to aneugenic agents, considerations on cancer risk evaluation are presented.

Zinc binding by the methylation signaling domain of the Escherichia coli Ada protein.

The Escherichia coli Ada protein repairs O6-methylguanine residues and methyl phosphotriesters in DNA by direct transfer of the methyl group to a cysteine residue located in its C- or N-terminal domain, respectively. Methyl transfer to the N-terminal domain causes it to acquire a sequence-specific DNA binding activity, which directs binding to the regulatory region of several methylation-resistance genes. In this paper we show that the N-terminal domain of Ada contains a high-affinity binding site for a single zinc atom, whereas the C-terminal domain is free of zinc. The metal-binding domain is apparently located within the first 92 amino acids of Ada, which contains four conserved cysteine residues. We propose that these four cysteines serve as the zinc ligand residues, coordinating the metal in a tetrahedral arrangement. One of the putative ligand residues, namely, Cys69, also serves as the acceptor site for a phosphotriester-derived methyl group. This raises the possibility that methylation-dependent ligand reorganization about the metal plays a role in the conformational switching mechanism that converts Ada from a non-sequence-specific to a sequence-specific DNA-binding protein.

Overproduction and dissection of proteins by the expression-cassette polymerase chain reaction.

We report an efficient, general approach for the construction of protein-overproducing strains of Escherichia coli. The method, expression-cassette polymerase chain reaction (ECPCR), allows the insertion of virtually any contiguous coding sequence between sequences that direct high-level protein biosynthesis in E. coli. The gene expression cassettes obtained by ECPCR are inserted into a regulated overexpression plasmid, and the resulting construct is used to transform E. coli. By effecting simultaneous 5' and 3' modification of a coding sequence, ECPCR permits the facile generation of mutant proteins having N- and/or C-terminal truncations. The method is a highly efficient way to dissect a multidomain protein into its component domains. The efficiency of the ECPCR approach is demonstrated in this study by construction of permuted overexpression vectors for the first two extracellular domains of the human CD4 protein.