Evaluation of procalcitonin in hemorrhagic shock: a pilot study.

Abstract: An increased secretion of procalcitonin (PCT) is primarily due to systemic inflammation of bacterial origin, as PCT is used to diagnose and manage sepsis. However, other conditions can induce high plasma levels of PCT, and hemorrhagic shock may be one of these as we found in clinical practice. The aim of this pilot, observational and prospective study was to investigate the role of PCT in hemorrhagic shock and if it could help in distinguishing between different types of shock. We enrolled 15 patients who entered the shock room of our Emergency Department (ED) with a diagnosis of hemodynamic shock, defined as hypotension (systolic blood pressure < 90 mmHg, or medial arterial pressure < 65 mmHg), and/or elevated lactate level (> 2 mmol/L), with one or more signs of cerebral or systemic hypoperfusion. For all the patients we dosed PCT at the time of admission, and we collected them into three different groups - septic, hemorrhagic and mixed shock - based on clinical presentation and laboratory and instrumental examination. First results did not show a significant increase of PCT in patients with hemorrhagic shock alone (average 0.12 ± 0.07 ng/mL), while PCT levels were similarly high in those with septic and mixed shock (17.63 ± 32.16 and 24.62 ± 33.02 respectively). PCT is not a marker of bleeding shock and does not help in distinguishing if bleeding or sepsis have the major impact on hemodynamics in those with mixed shock. However, patients with sepsis usually access the ED a few days after the initial infectious and inflammatory process has begun, while those with a major bleeding ask for intervention at the very first beginning. Thus, it may be helpful to see is PCT levels rise after some time from the bleeding start, or to investigate a different biomarker that rises earlier in course of systemic disfunction, such as presepsin. Finally, we also aimed at investigating if PCT levels would show any correlation with age of patients, regardless of the type of shock: results provided an higher PCT in individuals ≥ 80 years old, than in those < 80 years old.

Management of maxillary artery pseudoaneurysm in Emergency Department: a narrative review.

Abstract: Tooth extraction is a common procedure that is performed routinely and is associated with very few risks. The formation of a pseudoaneurysm as a direct result of tooth extraction has not been widely reported in published studies; it is more frequent as a complication of orthognathic surgery (1). The purpose of this paper is to describe the literature of maxillary artery pseudoaneurysm and its diagnosis and treatment in the Emer-gency Department. The search engine we used is Pubmed. 39 studies were analyzed; mainly, they were case reports. In this study, we will analyze the cases of pseudoaneurysm formation following dental extraction and orthognotia surgery which are reported in literature.

Mediterranean diet and the prevention of non-alcoholic fatty liver disease: results from a case-control study.

OBJECTIVE: Few studies report that Mediterranean dietary (MD) pattern has a beneficial role in the progression of non-alcoholic fatty liver disease (NAFLD). Evidence on its potential effect on the onset of disease are, however, scanty. With our study, we evaluated whether MD affects the risk of NAFLD with a large case-control study performed in Italy. PATIENTS AND METHODS: Three hundred and seventy-one cases of NAFLD and 444 controls were questioned on the demographic data and their dietary habits before diagnosis. Additionally, information about lifestyles and other related diseases, such as hypertension and diabetes mellitus were collected. The MD adherence was assessed using a pre-defined Mediterranean Diet Score (MDS). Odds ratios (OR) and 95% confidence intervals (CI) were obtained using a multiple logistic regression model. RESULTS: A high adherence to the MD is significantly associated with decreased risk of NAFLD (OR: 0.83 95% CI: 0.71-0.98). When the different MD components were examined separately, higher legumes consumption (OR: 0.62 95% CI: 0.38-0.99) and high fish consumption (OR 0.38 95% CI: 0.17-0.85) were reported to be protective against NAFLD. CONCLUSIONS: Our study shows that a high adherence to the MD decreases the risk of NAFLD.

Idiopathic sudden sensorineural hearing loss and ménière syndrome: The role of cerebral venous drainage.

OBJECTIVES: To evaluate the influence of cerebral venous drainage on the pathogenesis of idiopathic sudden sensorineural hearing loss (ISSHL) and Ménière syndrome (MD). DESIGN: Observational, prospective, cohort study. SETTING: ENT and Cardiology Departments (University of Bari, Policlinico Hospital, Bari, Italy). PARTICIPANTS: We enrolled 59 consecutive patients (32 males, mean age 53.05 + 15.37 years): 40 ISSHL and 19 MD. MAIN OUTCOME MEASURE: All patients underwent physical examination, biochemical evaluation (glycemic and lipid profile, viral serology, C reactive protein, etc), audiometric (tonal, vocal, vestibular evoked myogenic potentials and auditory brainstem response test) and impedentiometric examination. The pure tone average (PTA) was calculated for the following frequencies: 250, 500, 1000, 2000, 3000, 4000, 8000. An echo-color Doppler evaluation of the venous cerebral veins, internal jugular (IJV) and vertebral veins (VV) at supine and 90° position was performed. RESULTS: No morphological alterations were found both in patients and controls. There were no signs of stenosis, blocked flow, membranes, etc. We found lower minimum, mean and maximum velocities in distal IJVs (P = .019; P = .013; P = .022; respectively) and left VVs (P = .027; P = .008; P = .001; respectively) in supine (0°) position in both MD and ISSHL patients as compared to controls. The same was for orthostatic position (90°). We found negative correlations between the velocities in extracranial veins and PTA values: therefore, the worst the audiometric performance of the subjects, the lower the velocities in the venous cerebral drainage. CONCLUSIONS: Idiopathic sudden sensorineural hearing loss and Ménière syndrome patients showed altered venous flow in IJVs and VVs as compared to controls, independently from posture. This different behavior of venous tone control can influence the ear performance and may have a role in the pathogenesis of both diseases.

Goat casein genotypes are associated with milk production traits in the Sarda breed.

The aim of the current work was to analyze, in the Sarda breed goat, genetic polymorphism within the casein genes and to assess their influence on milk traits. Genetic variants at the CSN1S1, CSN2, CSN1S2 and CSN3 gene loci were investigated using PCR-based methods, cloning and sequencing. Strong alleles prevailed at the CSN1S1 gene locus and defective alleles also were revealed. Null alleles were evidenced at each calcium-sensitive gene locus. At the CSN3 gene locus, we observed a prevalence of the CSN3 A and B alleles; the occurrence of rare alleles such as CSN3 B'', C, C', D, E and M; and the CSN3 S allele (GenBank KF644565) described here for the first time in Capra hircus. Statistical analysis showed that all genes, except CSN3, significantly influenced milk traits. The CSN1S1 BB and AB genotypes were associated with the highest percentages of protein (4.41 and 4.40 respectively) and fat (5.26 and 5.34 respectively) (P < 0.001). A relevant finding was that CSN2 and CSN1S2 genotypes affected milk protein content and yield. The polymorphism of the CSN2 gene affected milk protein percentage with the highest values recorded in the CSN2 AA goats (4.35, at P < 0.001). The CSN1S2 AC goats provided the highest fat (51.02 g/day) and protein (41.42 g/day) (P < 0.01) production. This information can be incorporated into selection schemes for the Sarda breed goat.

Intracardiac Doppler assessment of left valve inflow in first-trimester fetuses with increased nuchal translucency: preliminary observations in trisomy 21.

OBJECTIVES: To perform a qualitative assessment of the pulsed Doppler waveform profile at the level of left atrioventricular valve inflow in first-trimester fetuses with increased nuchal translucency thickness (NT), in order to compare those with trisomy 21 and those with normal karyotype. METHODS: This was a review of 285 consecutive fetuses with increased NT. Pulsed Doppler velocity waveforms of left atrioventricular valve inflow were recorded. The E-wave, A-wave and velocity profile in the aorta were displayed. Cases were classified into two patterns: Pattern A included those in which the E-wave velocity crossed the A-wave before the baseline in all waveforms; Pattern B included those in which the lowest E-wave velocity crossed the baseline but not the A-wave in at least one of the profiles. The karyotype was determined and the frequency of occurrence of Patterns A or B in fetuses with normal karyotype and those with trisomy 21 were compared. RESULTS: Of the 285 cases, 230 were assigned to Pattern A and 55 to Pattern B. There were 47 cases of trisomy 21, 22 had other chromosomal abnormalities, and 212 had a normal karyotype; in four cases the karyotype was unknown. Among the 212 karyotypically normal fetuses, five had heart defects, five had other structural defects, three suffered spontaneous intrauterine death and one was terminated. Pattern A was found in 200/212 (94.3%) cases with normal karyotype, in 12/47 (25.5%) cases with trisomy 21, and in 17/22 (77.3%) cases with other chromosomal abnormalities. Pattern B was found in 12/212 (5.7%) cases with normal karyotype, in 35/47 (74.5%) cases with trisomy 21 (chi-square test, P < 0.001), and in 5/22 (22.7%) cases with other chromosomal abnormalities. CONCLUSIONS: Intracardiac Doppler qualitative assessment of left valve inflow in first-trimester fetuses with increased NT shows differences between normal and trisomy 21 fetuses, probably reflecting differences in myocardial function.

In vitro selection of HIV and CMV specific T-lymphocytes.

CMV and HIV produce life-long infections. During CMV infection, cellular responses mediated by virus specific CD8 and CD4 lymphocytes are effective, while during HIV infection cellular responses are ineffective in the long run. In recent years, much work has been carried out to better characterize such responses by using different methodologies to define the fine epitope specificity, the frequency and the function of specific T-cells. These studies have diagnostic and therapeutic implications. In fact, monitoring of specific lymphocytes may help define the immune status of the patients for therapeutic interventions. Identification of CD8 and CD4 epitopes allows the use of relevant peptides for lymphocyte stimulation or for vaccine development. Enumeration of specific cells permits a quantitative estimate of the immune response. In vitro selection provides large numbers of virus specific T-cells for studies on clonal composition, on epitope mapping and on HLA restriction as well as for therapeutic immunoreconstitution with ex vivo expanded T-cells.

Preservation of clonal heterogeneity of the Pneumocystis carinii-specific CD4 T cell repertoire in HIV infected, asymptomatic individuals.

The loss of CD4 lymphocytes in HIV disease associates with opportunistic infections. Since diverse CD4 T cell clones respond to an opportunistic pathogen, we asked whether CD4 depletion deletes selected clones in the repertoire (vertical depletion) or it affects all clones by reducing the cell number in each progeny without affecting the overall number of clones (horizontal depletion). Understanding this point may help explain the mode of CD4 depletion and the mode of immunoreconstitution after therapy. Therefore we examined the CD4 T cell repertoire specific for Pneumocystis carinii, a relevant opportunistic pathogen in AIDS, in HIV-infected, asymptomatic individuals. We identified two patients of 36 asymptomatics for lack of proliferation to P. carinii, suggesting selective depletion of specific CD4 cells. To investigate clonal heterogeneity of P. carinii-responsive CD4 lymphocytes, specific CD4 T cell lines were generated and studied by TCR BV gene family usage and CDR3 length analysis (spectratyping). Clonal heterogeneity was similar in antigen-specific CD4 lines generated from P. carinii non-responding HIV seropositives and from controls. Thus, despite undetectable response to the pathogen, residual specific cells probably prevent overt infection and, when expanded in vitro, exhibit a clonal diversity similar to normal controls. These findings suggest a horizontal, rather than vertical, depletion in these asymptomatic patients.

Analysis of the antigen specific T cell repertoires in HIV infection.

In addition to HIV infection, several acquired immunodeficiencies lead to depletion of CD4 lymphocytes. These include immunosuppression resulting from high dose cancer chemotherapy or induced to control graft rejection, as well as in autoimmune diseases. The consequence of this depletion is an increased susceptibility to opportunistic infections or the inability to control primary infection in the case of HIV infection. In all instances a full or partial immunoreconstitution is desirable. In order to monitor the cellular immune state of a patient, rational information cannot be simply derived from phenotypic quantification of T lymphocytes. Instead loss or recovery of CD4 cells should be monitored by defining the specificity, the function and the clonality of the relevant cell population. Several methods are now available for this type of investigation. Here we describe an approach for the definition of clonal heterogeneity of antigen specific CD4 lymphocytes, a parameter that may help monitor loss or reconstitution in acquired immunodeficiencies. As examples of antigen specific CD4 T cell responses we focused on Pneumocystis carinii and on cytomegalovirus, as prototypic opportunistic pathogens which are responsible for severe infections in AIDS and in other immunosuppressive conditions which arise for instance following transplantation. Specific CD4 T cell lines were generated from normal controls and from seropositives in order to select antigen specific lymphocytes. The cells were subsequently analyzed for clonal diversity according to TCR BV gene family usage and according to TCR CDR3 size heterogeneity (spectratyping).

Epitope focus, clonal composition and Th1 phenotype of the human CD4 response to the secretory mycobacterial antigen Ag85.

Lymphoproliferation of healthy donors was tested against mycobacterial antigens (PPD, Ag85, Ag85 peptides). All PPD responders recognized the secretory antigen Ag85 and the peptide specificity for Ag85B was defined. Peptide 91-108 was recognized by 85% of donors. In addition, all CD4 T cell lines generated from 12 donors against PPD or Ag85 responded to 91-108. When this peptide was used to generate T cell lines, the cells responded also to tuberculins from atypical mycobacterial species. Thus the cross-reactive peptide behaved as quasi-universal. The analysis of TCR-BV gene usage by cell lines showed that most Ag85-specific T cells correspond to 91-108-specific clonotypes. Intracytoplasmic staining of cell lines after phorbol myristate acetate stimulation resulted in dominance of interferon-gamma (IFN-gamma)-IL-4 double-positive cells, whereas antigen stimulation resulted in production of IFN-gamma only. The data show that peptide 91-108 is the major focus of the CD4 response to mycobacterial antigens in peripheral blood mononuclear cells and in T cell lines from PPD responders.

Mucosal exposure to subinfectious doses of SIV primes gut-associated antibody-secreting cells and T cells: lack of enhancement by nonneutralizing antibody.

It has been suggested that the presence of immunoglobulin and complement receptors on rectal epithelium may facilitate the entry of HIV complexed to nonneutralizing antibody. We tested this hypothesis using simian immunodeficiency virus (SIV) infection of rhesus macaques. First, in a pilot study, a nonneutralizing IgG fraction of macaque anti-SIV gp120 was shown to enhance the immunogenicity of SIV envelope following rectal immunization. The same antibody was then mixed with a subinfectious dose of SIV and the occurrence of rectal infection was compared with virus alone. Animals were not infected overtly and were rechallenged with a 10-fold higher dose of virus with and without addition of antibody. There was no evidence of antibody-mediated infection, since equal numbers of macaques became infected, regardless of the presence of antibody. In addition, the application of immune complexes did not alter significantly the subsequent virus load or the immune responses generated. In seronegative animals, in which virus and proviral DNA were undetectable in PBMC and tissues, SIV-specific T-cell responses and antibody-secreting cells were found in systemic and gut-associated sites. Our results show that nonneutralizing antibody neither facilitated nor enhanced rectal infection with SIV, in the small number of animals used, despite the consistent trend for this antibody to enhance antibody responses to gp120 following rectal immunization with immune-complexed antigen. However, mucosal exposure to subinfectious doses of virus primed both systemic and local immunity, regardless of addition of nonneutralizing antibody.

Genetically modified immunocompetent cells in HIV infection.

Even in the era of highly active antiretroviral therapy (HAART), gene therapy (GT) can remain a promising approach for suppressing HIV infection, especially if complemented with other forms of pharmacological and immunological intervention. A large number of vectors and targets have been studied. Here we discuss the potential of genetically treated, antigen-specific immunocompetent cells for adoptive autologous immunotherapy of HIV infection. Cellular therapies with gene-modified CD8 and CD4 lymphocytes are aimed at reconstituting the antigen-specific repertoires that may be deranged as a consequence of HIV infection. Even if complete eradication of HIV from the reservoirs cannot be achieved, reconstitution of cellular immunity specific for opportunistic pathogens and for HIV itself is a desirable option to control progression of HIV infection and AIDS pathogenesis better.

Cytokine gene expression and T-cell proliferative responses in lymph node mononuclear cells from children with early stage human immunodeficiency virus infection.

BACKGROUND AND OBJECTIVES: The immunologic events taking place in secondary lymphoid tissue from children with early stage human immunodeficiency virus (HIV) infection are poorly understood. The aim of this study was to investigate cytokine gene expression and proliferative responses in lymph node (LN) biopsies from five children with early stage HIV infection, in the context of LN morphology and viral load. DESIGN AND METHODS: The design of the study was approved by the local Ethical Committee. Cytokine gene expression was studied in LN biopsies and in paired peripheral blood (PB) samples from HIV-infected children by reverse transcriptase-polymerase chain reaction. T-cell proliferation was assessed by 3H-thymidine incorporation. Viral burden in germinal centers was assessed by video densitometric analysis following immunohistochemical staining for HIV p24. RESULTS: Interleukin (IL)-2, IL-4 and IL-5 mRNA were not detected in any LN or PB sample from HIV-infected children. Interferon (IFN)-gamma mRNA was found only in CD8+ cells. IL-12 p35, IL-10, transforming growth factor-(TGF)-beta1, regulated on activation normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and IL-16 transcripts were detected in all samples. Proliferation of LN and PB mononuclear cells to polyclonal mitogens and soluble (recall and HIV-related) antigens was impaired as compared with the responses in a group of age-matched healthy controls. INTERPRETATION AND CONCLUSIONS: Changes in cytokine gene expression and T-cell proliferative responses are already detectable in lymph nodes from HIV-infected children at an early stage of disease.

Cd4(+) T cell response to Leishmania spp. in non-infected individuals.

T cell mediated immunity is known to play a central role in the host response to control intra-cellular pathogens. This work demonstrates the presence of specific CD4(+) T cells to Leishmania spp. antigens in peripheral mononuclear cells of naïve individuals (normal volunteers from non-endemic regions). The responder population was expanded by generation of antigen-specific T cell lines, which were produced by repeated stimulation with fixed promastigotes and autologous irradiated PBMC as antigen presenting cells. The leishmania-T cell lines were shown to proliferate in response to different species of the parasite (L. amazonensis, L. braziliensis, and L. donovani), but not to other recall antigens such as Candida albicans or tetanus toxoid. A preferential expansion of IFNgamma and IL-2 producing Th1-like T cells was observed. The leishmania-reactive cells were distributed between CD4(+) CD45RA(+) ("naïve") and CD4(+) CD45R0(+) ("memory") populations. Although limiting dilution analysis showed a precursor frequency 3 times lower within the naïve compartment, similar numbers of T cell lines were derived from both purified subpopulations. This study using leishmania-specific CD4(+) T cell lines produced from normal individuals should provide information on cellular immune responses that are triggered by the parasite and how infection impacts the naïve T cell repertoire.

Natural analogue peptides of an HIV-1 GP120 T-helper epitope antagonize response of GP120-specific human CD4 T-cell clones.

Neutralizing antibodies and specific cytotoxic T lymphocytes (CTL) may contribute to controlling viral spread, and ideally, to virus clearance in HIV infection. Both effector mechanisms depend on specific CD4 T-helper (Th) cells. Nevertheless, HIV hypervariability facilitates appearance of escape mutants for antibodies and for CTL responses. Here we also show that natural mutations (i.e., from sequences of different HIV strains) in an immunodominant Th epitope recognized by human CD4 clones specific for the envelope glycoprotein gp120 escape CD4 T-cell recognition. Furthermore, several natural analogue peptides exert an antagonistic function by inhibiting proliferative response of T cells specific to gp120 with a wild-type sequence. If similar events occur in vivo, they may represent an additional escape mechanism for HIV. In fact, antagonism for CD4 Th response may occur during superinfection with a different strain, or with the appearance of a variant carrying a mutated antagonistic sequence. In both cases, impaired Th cell function could lead to reduced immune control of HIV infection by interfering with CTL and antibody response.

Cytogenetic response to autografting in chronic myelogenous leukemia correlates with the amount of BCR-ABL positive cells in the graft.

OBJECTIVE: An important step in successful autografting of patients with chronic myelogenous leukemia is the delivery of a leukemia-free graft. We conducted this study to determine whether the cytogenetic response after autografting was correlated with the number of BCR ABL-positive cells present within the stem cell grafts. MATERIALS AND METHODS: By BCR-ABL mRNA quantification, we studied the serial pheresis products from 40 Philadelphia (Ph)-positive patients who received ICE/mini-ICE mobilization therapy and underwent autologous stem cell transplantation. We correlated the residual disease within the graft reinfused with the cytogenetic response following transplantation, taking into consideration those responses that lasted 12 months or more. RESULTS: Thirty-two patients received a graft with 0-35% Ph-metaphases and 19 received a graft with BCR-ABL/ABL ratio < or =0.01. After a median of 27 months (range, 12-50) from transplant, 18 patients achieved complete or major cytogenetic response lasting at least 12 months, and 14 of them (78%) received a graft with BCR-ABL/ABL ratio < or =0.01 (range, 0.0003-0.01). Twenty-two patients experienced short-lived responses or had >35% Ph-positive cells in the marrow after transplant, but only 5 of them (23%) had a graft with BCR-ABL/ABL ratio < or =0.01 (range, 0.001-0.01). Therefore, we found a strong association between a BCR-ABL/ABL ratio less than or =0.01 and the achievement of complete or major cytogenetic remission after autografting (chi(2) test, p = 0.0001). Patients reinfused with grafts contaminated at low levels with leukemic cells also showed a longer duration of the response (log-rank test, p = 0.0009). Eleven patients were reinfused with the lowest level of contaminated stem cell collections, according to the BCR-ABL/ABL ratio. None of these patients experienced prolonged neutropenia or thrombocytopenia following stem cell reinfusion and nine of them had long-lasting complete or major cytogenetic responses after transplant. CONCLUSION: This study demonstrates that the number of BCR-ABL positive cells present in a stem cell graft is an important predictive factor for the achievement and the duration of cytogenetic response after autografting. [corrected]

HIV-1 Tat mutants in the cysteine-rich region downregulate HLA class II expression in T lymphocytic and macrophage cell lines.

Human macrophage and T cell lines were stably transfected with HIV-1 wild-type Tat or Tat mutants in the cysteine-rich region displaying trans-dominant negative effects on HIV-1 life cycle. The expression of HLA class I and class II molecules was not affected by wild-type Tat. Tat mutants, instead, profoundly down-regulated in a dose-dependent fashion the expression of class II, but not of class I, in both cell types by acting at the transcriptional level. Down-regulation was manifested on constitutive and IFN-gamma-induced class II gene expression and did not correlate with reduced transcription of the AIR-1 gene product CIITA, the major transcriptional activator of class II genes, indicating that Tat mutants did not act by inhibiting AIR-1 gene expression. Class II down-modulation had important functional implications in macrophages, as both antigen processing and presenting capacity were inhibited. These results represent the first evidence that a modified HIV-1 Tat product can act as a potent immunosuppressor by inhibiting the HLA class II expression necessary for triggering both cellular and humoral responses against pathogens. The use of these HIV-1 Tat mutants also discloses new opportunities to investigate the molecular mechanisms underlying the coordinate HLA class II gene transcription.

Quantitative competitive reverse transcriptase-polymerase chain reaction for BCR-ABL on Philadelphia-negative leukaphereses allows the selection of low-contaminated peripheral blood progenitor cells for autografting in chronic myelogenous leukemia.

The Philadelphia (Ph) translocation t(9;22) results in the creation of the BCR-ABL gene, which is now regarded as central to the mechanism that underlies the chronic phase of chronic myelogenous leukemia (CML). From a clinical point of view, BCR-ABL mRNA detection has become the basis for the study of minimal residual disease in CML, particularly when a complete cytogenetic remission is achieved after interferon-alpha (IFN-alpha) therapy or allogeneic stem cell transplantation. We have recently demonstrated that it is possible to mobilize normal peripheral blood progenitor cells (PBPC) in higher rates if this procedure is performed during the early chronic phase. In an attempt to monitor the leukemic cell content of PBPC collections, we used quantitative-competitive RT-PCR (QC-RT-PCR). Thirty consecutive Philadelphia (Ph) chromosome positive patients were enrolled in this study. After chemotherapy and G-CSF, 14 patients achieved 100% Ph-negative metaphases, nine patients had < or =34% and seven patients >34% leukemic metaphases. A total of 116 collection samples were studied. For each sample, BCR-ABL transcript numbers and BCR-ABL/ABL ratio were evaluated. A highly significant correlation between Ph-positive metaphases and BCR-ABL transcript numbers (r = 0.84, P < 0.0001) or BCR-ABL/ABL ratio (r = 0.86, P < 0.0001) was found. For patients that underwent the procedure in early chronic phase, Ph-negative collections showed different levels of BCR-ABL expression. BCR-ABL transcript numbers varied from a median of 100/microg RNA in the first and second leukaphereses, to 500/microg RNA in the third and fourth leukaphereses, and 1500/microg RNA in the fifth leukapheresis (P = 0.002). BCR-ABL/ABL ratio values showed similar kinetics. We have also demonstrated that there is a correlation between low values in BCR-ABL/ABL ratio (< or =0.01) in the reinfused PBPC and the achievement of cytogenetic remission after autografting (chi2 test, P = 0.01). In conclusion, this study demonstrates that QC-RT-PCR for BCR-ABL is a reliable and helpful method for monitoring residual leukemic load in mobilized PBPC, particularly in Ph-negative collections. Moreover, QC-RT-PCR allows selection of the best available collections for reinfusion into patients after myeloablative therapy.

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.