Global transcriptomic changes occur in uterine fluid-derived extracellular vesicles during the endometrial window for embryo implantation.

STUDY QUESTION: Are uterine fluid-derived extracellular vesicles (UF-EVs) a 'liquid biopsy' reservoir of biomarkers for real-time monitoring of endometrial status? SUMMARY ANSWER: The transcriptomic cargo of UF-EVs reflects the RNA profile of the endometrial tissue as well as changes between the non-receptive and the receptive phase, possibly supporting its use for a novel endometrial receptivity test. WHAT IS KNOWN ALREADY: EVs have been previously isolated from uterine fluid, where they likely contribute to the embryo-endometrium crosstalk during implantation. Based on a meta-analysis of studies on endometrial tissue implantation-associated genes and the human exosomes database, 28 of the 57 transcripts considered as receptivity markers refer to proteins present in human exosomes. However, the specific transcriptomic content of receptive phase UF-EVs has yet to be defined. STUDY DESIGN, SIZE, DURATION: Two experimental series were set up. First, we simultaneously sequenced RNA species derived from paired UF-EVs and endometrial tissue samples collected from physiologically cycling women. Second, we analyzed RNA species of UF-EVs collected during the non-receptive (LH + 2) and receptive (LH + 7) phase of proven fertile women and from the receptive (LH + 7) phase of a population of women undergoing ART and transfer of euploid blastocysts. PARTICIPANTS/MATERIALS, SETTING, METHODS: For paired UF-endometrial tissue sampling, endometrial tissue biopsies were obtained with the use of a Pipelle immediately after UF collection performed by lavage of the endometrial cavity. Overall, n = 87 UF samples were collected and fresh-processed for EV isolation and total RNA extraction, while western blotting was used to confirm the expression of EV protein markers of the isolated vesicles. Physical characterization of UF-EVs was performed by Nanoparticle Tracking Analysis. To define the transcriptomic cargo of UF-EV samples, RNA-seq libraries were successfully prepared from n = 83 UF-EVs samples and analyzed by RNA-seq analysis. Differential gene expression (DGE) analysis was used to compare RNA-seq results between different groups of samples. Functional enrichment analysis was performed by gene set enrichment analysis with g:Profiler. Pre-ranked gene set enrichment analysis (GSEA) with WebGestalt was used to compare RNA-seq results with the gene-set evaluated in a commercially available endometrial receptivity array. MAIN RESULTS AND THE ROLE OF CHANCE: A highly significant correlation was found between transcriptional profiles of endometrial biopsies and pairwise UF-EV samples (Pearson's r = 0.70 P < 0.0001; Spearman's ρ = 0.65 P < 0.0001). In UF-EVs from fertile controls, 942 gene transcripts were more abundant and 1305 transcripts less abundant in the LH + 7 receptive versus the LH + 2 non-receptive phase. GSEA performed to evaluate concordance in transcriptional profile between the n = 238 genes included in the commercially available endometrial receptivity array and the LH + 7 versus LH + 2 UF-EV comparison demonstrated an extremely significant and consistent enrichment, with a normalized enrichment score (NES)=9.38 (P < 0.001) for transcripts up-regulated in LH + 7 in the commercial array and enriched in LH + 7 UF-EVs, and a NES = -5.40 (P < 0.001) for transcripts down-regulated in LH + 7 in the commercial array and depleted in LH + 7 UF-EVs. When analyzing LH + 7 UF-EVs of patients with successful versus failed implantation after transfer of one euploid blastocyst in the following cycle, we found 97 genes whose transcript levels were increased and 64 genes whose transcript levels were decreased in the group of women who achieved a pregnancy. GSEA performed to evaluate concordance in transcriptional profile between the commercially available endometrial receptivity array genes and the comparison of LH + 7 UF-EVs of women with successful versus failed implantation, demonstrated a significant enrichment with a NES = 2.14 (P = 0.001) for transcripts up-regulated in the commercial array in the receptive phase and enriched in UF-EVs of women who conceived, and a not significant NES = -1.18 (P = 0.3) for transcripts down-regulated in the commercial array and depleted in UF-EVs. In terms of physical features, UF-EVs showed a homogeneity among the different groups analyzed except for a slight but significant difference in EV size, being smaller in women with a successful implantation compared to patients who failed to conceive after euploid blastocyst transfer (mean diameter ± SD 205.5± 22.97 nm vs 221.5 ± 20.57 nm, respectively, P = 0.014). LARGE SCALE DATA: Transcriptomic data were deposited in NCBI Gene Expression Omnibus (GEO) and can be retrieved using GEO series accession number: GSE158958. LIMITATIONS, REASONS FOR CAUTION: Separation of RNA species associated with EV membranes might have been incomplete, and membrane-bound RNA species-rather than the internal RNA content of EVs-might have contributed to our RNA-seq results. Also, we cannot definitely distinguish the relative contribution of exosomes, microvesicles and apoptotic bodies to our findings. When considering patients undergoing ART, we did not collect UFs in the same cycle of the euploid embryo transfer but in the one immediately preceding. We considered this approach as the most appropriate in relation to the novel, explorative nature of our study. Based on our results, a validation of UF-EV RNA-seq analyses in the same cycle in which embryo transfer is performed could be hypothesized. WIDER IMPLICATIONS OF THE FINDINGS: On the largest sample size of human EVs ever analyzed with RNA-seq, this study establishes a gene signature to use for less-invasive endometrial receptivity tests. This report is indeed the first to show that the transcriptome of UF-EVs correlates with the endometrial tissue transcriptome, that RNA signatures in UF-EVs change with endometrial status, and that UF-EVs could serve as a reservoir for potential less-invasive collection of receptivity markers. This article thus represents a step forward in the design of less-invasive approaches for real-time monitoring of endometrial status, necessary for advancing the field of reproductive medicine. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by a competitive grant from European Society of Human Reproduction and Embryology (ESHRE Research Grant 2016-1). The authors have no financial or non-financial competing interests to disclose. TRIAL REGISTRATION NUMBER: NA.

Embryonic extracellular vesicles as informers to the immune cells at the maternal-fetal interface.

Extracellular vesicle (EV) exchange is emerging as a novel method of communication at the maternal-fetal interface. The presence of the EVs has been demonstrated in the preimplantation embryo culture medium from different species, such as bovines, porcines and humans. Preimplantation embryo-derived EVs have been shown to carry molecules potentially able to modulate the local endometrial immune system. The non-classical major histocompatibility complex (MHC) class I molecule human leucocyte antigen (HLA)-G, the immunomodulatory molecule progesterone-induced blocking factor and some regulatory miRNAs species are contained in embryo-derived EV cargo. The implanted syncytiotrophoblasts are also well known to secrete EVs, with microvesicles exerting a mainly proinflammatory effect while exosomes in general mediate local immunotolerance. This review focuses on the current knowledge on the potential role of EVs released by the embryo in the first weeks of pregnancy on the maternal immune cells. Collectively, the data warrant further exploration of the dialogue between the mother and the embryo via EVs.

MTHFR, TS and XRCC1 genetic variants may affect survival in patients with myelodysplastic syndromes treated with supportive care or azacitidine.

We evaluated the impact of genomic polymorphisms in folate-metabolizing, DNA synthesis and DNA repair enzymes on the clinical outcome of 108 patients with myelodysplastic syndromes (MDS) receiving best supportive care (BSC) or azacitidine. A statistically significant association between methylenetetrahydrofolate reductase (MTHFR) 677T/T, thymidylate synthase (TS) 5'-untranslated region (UTR) 3RG, TS 3'-UTR -6 bp/-6 bp, XRCC1 399G/G genotypes and short survival was found in patients receiving BSC by multivariate analysis (P<0.001; P=0.026; P=0.058; P=0.024). MTHFR 677T/T, TS 3'-UTR -6 bp/-6 bp and XRCC1 399G/G genotypes were associated with short survival in patients receiving azacitidine by multivariate analysis (P<0.001; P=0.004; P=0.002). We then performed an exploratory analysis to evaluate the effect of the simultaneous presence of multiple adverse variant genotypes. Interestingly, patients with ⩾1 adverse genetic variants had a short survival, independently from their International Prognostic Scoring System (IPSS) and therapy received. To our knowledge, this is the first study showing that polymorphisms in folate-metabolizing pathway, DNA synthesis and DNA repair genes could influence survival of MDS patients.

Time to redefine endometriosis including its pro-fibrotic nature.

Endometriosis is currently defined as presence of endometrial epithelial and stromal cells at ectopic sites. This simple and straightforward definition has served us well since its original introduction. However, with advances in disease knowledge, endometrial stromal and glands have been shown to represent only a minor component of endometriotic lesions and they are often absent in some disease forms. In rectovaginal nodules, the glandular epithelium is often not surrounded by stroma and frequently no epithelium can be identified in the wall of ovarian endometriomas. On the other hand, a smooth muscle component and fibrosis represent consistent features of all disease forms. Based on these observations, we believe that the definition of endometriosis should be reconsidered and reworded as 'A fibrotic condition in which endometrial stroma and epithelium can be identified'. The main reasons for this change are: (1) to foster the evaluation of fibrosis in studies on endometriosis pathogenesis using animal models; (2) to limit potential false negative diagnoses if pathologists stick stringently to the current definition of endometriosis requiring the demonstration of endometrial stromal and glands; (3) to consider fibrosis as a potential target for treatment in endometriosis. This opinion article is aimed at boosting the attention paid to a largely neglected aspect of the disease. We hope that targeting the fibrotic process might increase success in developing new therapeutic approaches.

Glycolysis gene expression analysis and selective metabolic advantage in the clinical progression of colorectal cancer.

Production of lactate even in the presence of sufficient levels of oxygen (aerobic glycolysis) seems the prevalent energy metabolism pathway in cancer cells. The analysis of altered expression of effectors causing redirection of glucose metabolism would help to characterize this phenomenon with possible therapeutic implications. We analyzed mRNA expression of the key enzymes involved in aerobic glycolysis in normal mucosa (NM), primary tumor (PT) and liver metastasis (LM) of colorectal cancer (CRC) patients (pts) who underwent primary tumor surgery and liver metastasectomy. Tissues of 48 CRC pts were analyzed by RT-qPCR for mRNA expression of the following genes: hexokinase-1 (HK-1) and 2 (HK-2), embryonic pyruvate kinase (PKM-2), lactate dehydrogenase-A (LDH-A), glucose transporter-1 (GLUT-1), voltage-dependent anion-selective channel protein-1 (VDAC-1). Differences in the expression of the candidate genes between tissues and associations with clinical/pathologic features were studied. GLUT-1, LDH-A, HK-1, PKM-2 and VDAC-1 mRNA expression levels were significantly higher in PT/LM tissues compared with NM. There was a trend for higher expression of these genes in LM compared with PT tissues, but differences were statistically significant for LDH-A expression only. RAS mutation-positive disease was associated with high GLUT-1 mRNA expression levels only. Right-sided colon tumors showed significantly higher GLUT-1, PKM-2 and LDH-A mRNA expression levels. High glycolytic profile was significantly associated with poor prognosis in 20 metastatic, RAS-mutated pts treated with first-line chemotherapy plus Bevacizumab. Altered expression of effectors associated with upregulated glucose uptake and aerobic glycolysis occurs in CRC tissues. Additional analyses are warranted for addressing the role of these changes in anti-angiogenic resistance and for developing novel therapeutics.

Characterization of Brachyspira hyodysenteriae isolates from Italy by multilocus sequence typing and multiple locus variable number tandem repeat analysis.

AIMS: To evaluate and compare the capabilities of multilocus sequence typing (MLST) and multiple locus variable number tandem repeat analysis (MLVA) techniques to characterize Brachyspira hyodysenteriae isolates and to investigate the relationship between pleuromutilin resistance and genetic variability. METHODS AND RESULTS: MLST genotyping was performed on 180 B. hyodysenteriae isolates, and the results were evaluated considering profiles from 108 other strains previously reported in the database. In total, 37 sequence types were obtained. The MLVA approach completely characterized 172 strains and grouped the isolates into 22 different profiles. The combination of MLST and MLVA showed a slight increase in the discriminatory power, identifying 33 joint profiles. An antibiotic resistance analysis showed a reduction in the susceptibility to pleuromutilins over time, and a weak association between susceptibility to valnemulin and inclusion in clonal complex 4. CONCLUSION: MLST and MLVA are reliable methods for characterizing B. hyodysenteriae strains and they have comparable discriminatory power. SIGNIFICANCE AND IMPACT OF THE STUDY: The genotyping of B. hyodysenteriae isolates and a database of all the genetic profiles collected during the diagnostic activities could support traditional epidemiological investigations in identifying infection sources and routes of transmission among herds, and in developing more effective control measures.

Impact of salbutamol on muscle metabolism assessed by ³¹P NMR spectroscopy.

The potential ergogenic effects of oral salbutamol intake were demonstrated for decades but the underlying mechanisms remain to elucidate. We hypothesized that improved exercise performance after acute oral salbutamol administration is associated with changes in muscle metabolism. Twelve healthy, nonasthmatic, moderately trained, male subjects were recruited to compare in a double-blind crossover randomized study, an oral dose of salbutamol (4 mg) and a placebo. After treatment administration, subjects performed repetitive plantar flexions to exhaustion in a 3T magnet. Continuous (31) P nuclear magnetic resonance spectroscopy assessment of the calf muscles was performed at rest, during exercise, and during recovery. No significant difference between treatments was detected in metabolite concentration at rest (P > 0.05). Creatine phosphate and inorganic phosphate changes during and immediately after exercise were similar between treatments (P > 0.05). Intramuscular pH (pHi) was significantly higher at rest, at submaximal exercise but not at exhaustion with salbutamol (pHi at 50% of exercise duration, 6.8 ± 0.1/6.9 ± 0.1 for placebo and salbutamol, respectively, P < 0.05). The maximal power (28 ± 7 W/23 ± 7 W; P = 0.001) and total work (1702 ± 442 J/1381 ± 432 J; P = 0.003) performed during plantar flexions were significantly increased with salbutamol. Salbutamol induced significant improvement in calf muscle endurance with similar metabolic responses during exercise, except slight differences in pHi. Other mechanisms than changes in muscle metabolism may be responsible for the ergogenic effect of salbutamol administration.

Clinical impact of the HGF/MET pathway activation in patients with advanced gastric cancer treated with palliative chemotherapy.

In gastric cancer, available clinical studies focusing on the activated hepatocyte growth factor (HGF)/MET pathway are limited to surgical and often heterogeneous series. MET copy number gain (CNG) and an activating truncation in the HGF promoter (deoxyadenosine tract element, DATE+) were studied in tumors of 95 patients with advanced gastric cancer treated with palliative chemotherapy. Associations with overall survival (OS) and the pattern of metastatic disease were studied. Median OS was 9.7 months in 80 MET CNG <5 copies cases (MET-), and 6.4 months in 15 MET CNG was ⩾5 copies cases (MET+) (P=0.001). MET+ status confirmed the adverse prognostic effect in the multivariate model. A significantly different distribution of MET+/DATE+ and MET-/DATE- cases was observed between patients with and without peritoneal carcinomatosis (PC). MET+ status confirms its adverse prognostic role in advanced gastric cancer patients. The activated MET/HGF axis seems to be associated with PC. These findings are relevant to the development of anti-MET/HGF compounds.

Parallel-transmission-enabled magnetization-prepared rapid gradient-echo T1-weighted imaging of the human brain at 7 T.

One of the promises of Ultra High Field (UHF) MRI scanners is to bring finer spatial resolution in the human brain images due to an increased signal to noise ratio. However, at such field strengths, the spatial non-uniformity of the Radio Frequency (RF) transmit profiles challenges the applicability of most MRI sequences, where the signal and contrast levels strongly depend on the flip angle (FA) homogeneity. In particular, the MP-RAGE sequence, one of the most commonly employed 3D sequences to obtain T1-weighted anatomical images of the brain, is highly sensitive to these spatial variations. These cause deterioration in image quality and complicate subsequent image post-processing such as automated tissue segmentation at UHF. In this work, we evaluate the potential of parallel-transmission (pTx) to obtain high-quality MP-RAGE images of the human brain at 7 T. To this end, non-selective transmit-SENSE pulses were individually tailored for each of 8 subjects under study, and applied to an 8-channel transmit-array. Such RF pulses were designed both for the low-FA excitation train and the 180° inversion preparation involved in the sequence, both utilizing the recently introduced k(T)-point trajectory. The resulting images were compared with those obtained from the conventional method and from subject-specific RF-shimmed excitations. In addition, four of the volunteers were scanned at 3 T for benchmarking purposes (clinical setup without pTx). Subsequently, automated tissue classification was performed to provide a more quantitative measure of the final image quality. Results indicated that pTx could already significantly improve image quality at 7 T by adopting a suitable RF-Shim. Exploiting the full potential of the pTx-setup, the proposed k(T)-point method provided excellent inversion fidelity, comparable to what is commonly only achievable at 3 T with energy intensive adiabatic pulses. Furthermore, the cumulative energy deposition was simultaneously reduced by over 40% compared to the conventional adiabatic inversions. Regarding the low-FA k(T)-point based excitations, the FA uniformity achieved at 7 T surpassed what is typically obtained at 3 T. Subsequently, automated white and gray matter segmentation not only confirmed the expected improvements in image quality, but also suggests that care should be taken to properly account for the strong local susceptibility effects near cranial cavities. Overall, these findings indicate that the k(T)-point-based pTx solution is an excellent candidate for UHF 3D imaging, where patient safety is a major concern due to the increase of specific absorption rates.

kT -points: short three-dimensional tailored RF pulses for flip-angle homogenization over an extended volume.

With Transmit SENSE, we demonstrate the feasibility of uniformly exciting a volume such as the human brain at 7T through the use of an original minimalist transmit k-space coverage, referred to as "k(T) -points." Radio-frequency energy is deposited only at a limited number of k-space locations in the vicinity of the center to counteract transmit sensitivity inhomogeneities. The resulting nonselective pulses are short and need little energy compared to adiabatic or other B 1+-robust pulses available in the literature, making them good candidates for short-repetition time 3D sequences at high field. Experimental verification was performed on three human volunteers at 7T by means of an 8-channel transmit array system. On average, whereas the standard circularly polarized excitation resulted in a 33%-flip angle spread (standard deviation over mean) throughout the brain, and a static radio-frequency shim showed flip angle variations of 17% and up, application of k(T) -point-based excitations demonstrated excellent flip angle uniformity (8%) for a small target flip angle and with sub-millisecond durations.

Measuring perfusion and bioenergetics simultaneously in mouse skeletal muscle: a multiparametric functional-NMR approach.

A totally noninvasive set-up was developed for comprehensive NMR evaluation of mouse skeletal muscle function in vivo. Dynamic pulsed arterial spin labeling-NMRI perfusion and blood oxygenation level-dependent (BOLD) signal measurements were interleaved with (31)P NMRS to measure both vascular response and oxidative capacities during stimulated exercise and subsequent recovery. Force output was recorded with a dedicated ergometer. Twelve exercise bouts were performed. The perfusion, BOLD signal, pH and force-time integral were obtained from mouse legs for each exercise. All reached a steady state after the second exercise, justifying the pointwise summation of the last 10 exercises to compensate for the limited (31)P signal. In this way, a high temporal resolution of 2.5 s was achieved to provide a time constant for phosphocreatine (PCr) recovery (τ(PCr)). The higher signal-to-noise ratio improved the precision of τ(PCr) measurement [coefficient of variation (CV) = 16.5% vs CV = 49.2% for a single exercise at a resolution of 30 s]. Inter-animal summation confirmed that τ(PCr) was stable at steady state, but shorter (89.3 ± 8.6 s) than after the first exercise (148 s, p < 0.05). This novel experimental approach provides an assessment of muscle vascular response simultaneously to energetic function in vivo. Its pertinence was illustrated by observing the establishment of a metabolic steady state. This comprehensive tool offers new perspectives for the study of muscle pathology in mice models.

Inactivated Salmonella enterica serovar Typhimurium monophasic variant (S. Typhimurium 1,4,[5],12:i-) in sows is effective to control infection in piglets under field condition.

The monophasic variant of Salmonella enterica serovar Typhimurium, namely Salmonella 1,4,[5],12:i-, has been increasingly responsible for foodborne human cases of disease and is most frequently detected in pork, since the variant is widely spread in pig farms. The aim of this study was to assess the efficacy of an autologous vaccine in decreasing the prevalence of Salmonella 1,4,[5],12:i-, in pigs. The trial was performed in a multisite pig production system of Northern Italy. The autogenous vaccine was prepared from the Salmonella 1,4,[5],12:i- strain isolated from the clinical case occurring in the Farm. Different immunization protocols were applied, ranging from interventions only in sows or piglets, or both. Microbiological analysis was performed to assess faecal shedding in sows and their offspring from birth till end of the production cycle and organ colonization of slaughtered pigs. Body weight of pigs was recorded at different time-points. Humoral immune response was evaluated in serum samples of sows and piglets. S. Typhimurium 1,4,[5],12:i- determines reduction of animal growth and farm production, furthermore, contamination of carcasses at the slaughterhouse. The load of bacteria entering into the food processing chain is differently influenced by the regimen of administration of inactivated vaccine. In particular, a combined vaccination of sows and their offspring was able to improve the weight gain of growing pigs, to limit Salmonella colonization of organs and to reduce the number of carrier pigs, and hence lowering the risk of introducing Salmonella organisms in the slaughter process.

Functional MR evaluation of temporal and frontal language dominance compared with the Wada test.

OBJECTIVE: To evaluate the reliability of temporal and frontal functional MRI (fMRI) activation for the assessment of language dominance, as compared with the Wada test. PATIENTS AND METHODS: Ten patients with temporal lobe epilepsy were studied using blood oxygen level dependent fMRI and echoplanar imaging (1.5-T). Three tasks were used: semantic verbal fluency, covert sentence repetition, and story listening. Data were analyzed using pixel by pixel autocorrelation and cross-correlation. fMRI laterality indices were defined for several regions of interest as the ratio (L - R)/(L + R), L being the number of activated voxels in the left hemisphere and R in the right hemisphere. Wada laterality indices were defined as the difference in the percentages of errors in language tests between left and right carotid injections. RESULTS: Semantic verbal fluency: The asymmetry of frontal activation was correlated with Wada laterality indices. The strongest correlation was observed in the precentral/middle frontal gyrus/inferior frontal sulcus area. Story listening: The asymmetry of frontal, but not temporal, activation was correlated with Wada laterality indices. Covert sentence repetition: No correlation was observed. CONCLUSIONS: There was a good congruence between hemispheric dominance for language as assessed with the Wada test and fMRI laterality indices in the frontal but not in the temporal lobes. The story listening and the covert sentence repetition tasks increased the sensitivity of detection of posterior language sites that may be useful for brain lesion surgery.

A two-stage culture system for the induction of antibody-forming cell clones in cultures of normal human blood lymphocytes.

A two-stage culture method is described for the induction of a specific antibody response to sheep red cells (SRC) in microcultures at limiting dilutions of human peripheral blood lymphocytes (PBL). PBL from normal donors were cultured for 4 days with antigen and EBV using well defined conditions. The cells were then distributed in 10 microliter microcultures at different cell densities in order to estimate the frequency of responding units. The culture wells were tested for the presence of anti-SRC antibody by the spot test. The results show that the expression of antibody-forming cell clones in the second stage microcultures is strictly dependent on the presence of both antigen and EBV during the first stage cultures. The efficiency of the system was improved by the addition of 4% polyethylene glycol (PEG, MW 6000) in the first stage and its removal in the second stage and by the use of human serum (instead of fetal calf) in both stages. This approach permits the separation of different cellular events, occurring when human B cells are stimulated by antigen and represents a useful approach for studying the mechanisms of the specific immune response in man.

Induction of antibody forming cells with specificity for Candida albicans mannoproteins in cultures of human peripheral blood lymphocytes.

A method is described for the induction of a specific antibody response to Candida albicans in cultures of normal human peripheral blood lymphocytes (PBL). PBL were cultured in the presence of whole C. albicans cells and the antibody response was evaluated by the ELISPOT technique, on plastic wells coated with a purified candidal cell well mannoprotein (MP). Under the conditions described here, a specific antibody response was obtained in all of the eight donors tested. The response was antigen-dependent and antigen-specific, peaked around day 10-12 of culture and the antibodies belonged to both the IgM and the IgG isotypes. By testing the cultured cells on MP from different Candida species, the method permitted the detection of antibodies directed against MP epitopes shared by C. albicans and C. parapsilosis.

Diltiazem modulates monokine production in human mixed lymphocyte culture.

BACKGROUND: Calcium channel blockers are widely used in transplantation. Their immunosuppressive activity is well known and has been demonstrated both in vitro and in vivo. Nevertheless, their effect on cytokine production has never been reported. METHODS: One-way mixed lymphocyte cultures (MLCs) have been obtained from healthy human subjects. Cytokine production has been assessed by three different methods: by enzyme-linked immunosorbent assay on supernatants of MLC, by enzyme-linked immunospot method on MLC cells for measuring cytokine-producing cells, and by the reverse transcription polymerase chain reaction technique on MLC cells for measuring cytokine mRNAs. RESULTS: An interesting effect on proinflammatory monokines was observed: in this study, we demonstrate that the calcium antagonist diltiazem enhances interleukin-1beta and slightly reduces interleukin-6 production in MLC, but it has no effect on tumor necrosis factor-alpha levels. CONCLUSION: For the first time, a modulation of monokine production by diltiazem can be demonstrated. This evidence suggests that calcium antagonist drugs may exert effects on monocytes and possibly on other antigen-presenting cells.

The antigen-specific induction of normal human lymphocytes in vitro is down-regulated by a conserved HIV p24 epitope.

Synthetic peptides containing amino acid sequence 218-238 of the core protein p24 of human immunodeficiency virus type 1 (HIV-1) and progressively shorter sequences at its C-terminus, were tested for their effect on antigen dependent in vitro responses of peripheral blood lymphocytes (PBL) from normal human donors. A peptide as short as 7 amino acids, corresponding to a highly conserved sequence, was able to inhibit in a dose-dependent manner the induction of a specific primary antibody response to the sheep red cell (SRC) antigen, as well as the proliferative response to recall microbial antigens. The results of this study constitute additional evidence of the immunoinhibitory effects of HIV components and may help to unravel some of the pathogenic mechanisms of AIDS. Moreover, they are of potential relevance for the development of immunoprophylactic and therapeutic strategies.

Induction of a specific antibody response to Bordetella pertussis antigens in cultures of human peripheral blood mononuclear cells.

The role of specific antibodies in protective immunity to Bordetella pertussis has not yet been clearly defined. In the present work, the induction of a specific antibody response to B. pertussis in cultures of human peripheral blood mononuclear cells (PBMC) was investigated, on the assumption that the capacity of circulating lymphocytes to mount a specific response in vitro may provide a useful parameter for the evaluation of protective immunity. When PBMC from normal adult donors were cultured with a heat-inactivated B. pertussis whole-cell suspension, cells secreting antibodies to pertussis toxin, pertactin and filamentous haemagglutinin were generated consistently. The antibody response peaked between days 7 and 11 of culture and the antibodies produced were exclusively of the IgM class.

A mannoprotein constituent of Candida albicans cooperates with antigen in the induction of a specific primary antibody response in cultures of human lymphocytes.

The effects of Candida albicans mannoproteins on the induction of a primary antibody response to a T-dependent antigen, sheep erythrocytes (SRBC), in cultures of human blood lymphocytes, were investigated. Two experimental systems (bulk and limiting dilution cultures) allowing the detection of both enhancing and inhibitory effects, were used. In bulk cultures, antigen alone elicited a small number of specific antibody forming cells, unless IL-2 was supplied. Addition of the fungal mannoprotein extract or of a purified constituent of it increased 5 to more than 10 times the specific response. When limiting dilution analysis was performed, we observed that: a) a similar number of specific precursor cells was induced by antigen and either IL-2 or mannoprotein; b) the plot of the number of seeded cells versus the log of the fraction of negative cultures was linear in antigen and IL-2 triggered cultures but constantly deviated from linearity when the candidal stimulant was added. Thus, more than one type of precursor cell was limiting in these cultures, and the immunoenhancing effect of mannoprotein may involve multiple cellular interactions.

Immunoregulatory effect of a synthetic peptide corresponding to a region of protein p24 of HIV.

The effect of a synthetic peptide, corresponding to a sequence of HIV-1 p24 protein (amino acids 218-237), on in vitro immune responses was studied. The peptide inhibited in a dose-dependent manner the induction of an anti-SRC antibody response and of a PPD-specific proliferative response of human PBL. On the other hand, PHA-induced proliferation of human PBL and PPD-induced proliferation of a PPD-specific human T-cell line were not modified by comparable amounts of the peptide. These results suggest that structures from a protein (p24), present in the serum throughout the course of HIV infection, are able to interfere with the inductive stages of specific immune responses. These findings may help to unravel some of the pathogenic mechanisms of AIDS and may contribute to the development of vaccine strategies.