Identifying recent HIV infections using the avidity index and an automated enzyme immunoassay

We evaluated a procedure for identifying recent HIV infections, using sequential serum samples from 47 HIV-positive persons for whom the seroconversion date could be accurately estimated. Each serum sample was divided into two aliquots: one diluted with phosphate-buffered saline and the other diluted with 1 M guanidine. We assayed the aliquots with the automated AxSYM HIV1/2gO test (Abbott Diagnostics Division), without modifying the manufacturer's protocol. We then calculated the avidity index (AI): the ratio of the sample/cutoff value for the guanidine aliquot to that of the phosphate-buffered saline aliquot. We analyzed 216 serum samples: 34 samples were collected within 6 months of seroconversion (recent seroconversions), and 182 were collected after 6 months. The mean AIs, by time from seroconversion, were 0.68 +/- 0.16 (within 6 months) and 0.98 +/- 0.10 (after 6 months) (P < 0.0001). AI of <0.90 correctly identified 88.2% of recent infections but misclassified as recent infections 13.2% of serum samples collected afterward. The probability of an infection being classified as recent and having AI of > or = 0.90 would be 0.7% in a population with 5% recent infections. AI can identify with a certain degree of accuracy recent HIV infections, and being a quantitative index, it provides different levels of sensitivity and specificity, depending on the selected cutoff value. The standard assay procedure is not modified. This test is simple and inexpensive and could be used for surveillance, decision-making in treatment, and prevention.     

Four-color flow cytometric analysis of peripheral blood donor cell chimerism

Passenger leukocytes have been demonstrated to play significant roles in initiating and also regulating immune reactions after organ transplantation. Reliable techniques to detect donor leukocytes in recipients after organ transplantation are essential to analyze the role, function, and behavior of these leukocytes. In this report we describe a simple, reliable method to detect donor cells with low frequencies using peripheral blood samples. Detection of small numbers of major histocompatibility complex (MHC) mismatched cells was first studied using four-color flow cytometry in artificially created cell mixtures. By selecting the CD45(+) population and simultaneous staining with several leukocyte lineage markers (CD3, CD4, CD8, CD56, and CD19), MHC-mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood samples with a threshold sensitivity as low as 0.1%-0.2%. When the fresh peripheral blood mononuclear cells were separated by conventional Ficoll gradient purification, similar, but slightly lower levels of donor cells were detected. Blood samples obtained 1-5 months after liver, kidney, and intestine transplants revealed that the kind of organ allograft influenced levels and lineage pattern of the circulating donor cells. This procedure provided a simple and reliable method in determining early chimerism in transplant recipients. However, the detection of MHC-mismatched leukocytes of all lineages was much lower when frozen peripheral blood mononuclear cells were used.     

Thromboembolic diseases in neonates and children

Acquired and inherited prothrombotic risk factors increase the risk of thrombosis in neonates, infants and children. After suffering thrombosis white paediatric patients should be screened for common gene mutations, i.e. the factor V G1691A, factor II G20210A and MTHFR C677T genotypes, rare inherited prothromboticrisk factors, i.e. deficiencies of protein C,protein S, and antithrombin, plasminogen, probably inherited risk factors, i.e. fibrinogen, factor VIIIC, factor XII, new candidates, i.e. elevation of lipoprotein (a),and fasting homocysteine concentrations (3-6 months after thrombotic onset). Data interpretation is based on age-dependent reference ranges or the identification of causative gene mutations/polymorphisms with respect to individual ethnic backgrounds.     

Association of haplotypes in the beta-chemokine locus with multiple sclerosis

Linkage studies in multiple sclerosis (MS) identified several susceptibility loci. One of these regions includes chromosome 17q11 where a meta-analysis of data from three genome scans suggested linkage. This region encodes a cluster of genes for beta-chemokines or CC chemokine ligands (CCLs), which may be involved in the development of MS lesions. Here we aimed to test if CCL alleles and haplotypes are associated with MS. Using methods of linkage and association, we observed deviations from the expected 50% transmission of haplotypes from unaffected parents to their affected children at CCL2, CCL11-CCL8-CCL13 and CCL3 within the investigated 1.85 MB chromosomal segment. Analyses of the linkage disequilibrium map support that variants with possible relevance to MS can be located within these subregions. Identification of MS associated CCL variants may have direct clinical significance, as it can lead to the design of small competitive antagonists of these molecules with beneficial effects in the treatment of patients with early and active disease.     

Amygdala-prefrontal coupling depends on a genetic variation of the serotonin transporter

Major depression is conditionally linked to a polymorphism of the human serotonin transporter gene (SLC6A4). During the presentation of aversive, but not pleasant, pictures, healthy carriers of the SLC6A4 short (s) allele showed stronger activation of the amygdala on functional magnetic resonance imaging. s carriers also showed greater coupling between the amygdala and the ventromedial prefrontal cortex, which may contribute to the abnormally high activity in the amygdala and medial prefrontal cortex seen in major depression.     

Transient expression of keratin 19 is induced in originally negative interfollicular epidermal cells by adhesion of suspended cells

Keratin 19 and nuclear reactivity to an endogenous lectin, galectin-1, represent a potential marker of epidermal stem cells. We detected expression of keratin 19 and nuclear binding sites for galectin-1 in adult cells migrating from the hair follicle, where cells expressing keratin 19 are located in the bulge region. The results were compared with the expression of both markers in cells adhering from suspension prepared from the interfollicular epidermis without keratin-19-positive cells and with nuclear binding sites for galectin-1. The results were compared with data from basal cell carcinomas. All cells were analyzed concerning size, as it is known that cell diameter influences the clonogenic potential of keratinocytes. The major result of this study is the observation of transient expression of keratin 19 and nuclear galectin-1 binding sites in originally negative interfollicular epidermal cells induced by adhesion. These cells were very small in size, similar to basal cells of the interfollicular epidermis or the bulge region of the hair follicle. The influence of the suspension regimen on beta1-integrin expression, cell diameter and growth was also monitored. A population of cells highly positive for beta1 integrin of the same diameter as keratin-19-positive cells insensitive to induction of terminal differentiation by lack of anchorage was characterized. Cells of the same size were also observed in the keratin-19-positive cells of basal cell carcinomas. In conclusion, the expression of poor levels of differentiation induced by cell adhesion is transient. Also, keratin 19 expression should not be exclusively regarded as a marker of stem cell activity.     

Decreased phenylalanine uptake and turnover in patients with vitiligo

The human epidermis has the full machinery for autocrine L-phenylalanine turnover to L-tyrosine in keratinocytes and melanocytes. Phenylalanine hydroxylase (PAH) activities increase linearly with inherited skin colour (skin phototype I-VI, Fitzpatrick classification) yielding eightfold more activities in black skin compared to white skin. Moreover, UVB irradiation (1 MED) significantly increases epidermal PAH activities 24 h after exposure. Importantly, L-phenylalanine uptake and turnover in the pigment forming melanocytes is vital for initiation of melanogenesis. In this context it was shown that the uptake of this amino acid is regulated by calcium. The depigmentation disorder vitiligo provides a unique model to follow impaired L-phenylalanine turnover in the skin as well as in serum because affected individuals hold an impaired epidermal 6BH4 de novo synthesis/recycling and regulation including low epidermal PAH activities. After overnight fasting and oral loading with L-phenylalanine (100 mg/kg body weight), 29.6% of 970 patients tested (n=287/970) yielded serum phenylalanine/tyrosine ratios >or=4 and 35.3% (n=342/970) had mild to moderate hyperphenylalaninaemia (HPA), while 9.3% (n=90/970) had both serum L-phenylalanine levels >or=2.0 mg/dl and phe/tyr ratios >or=4.0. Isolated HPA was found in 26% (n=252/970), whereas 20.3% had only increased ratios (n=197/970). None of the patients had phenylketonuria and the family history for this metabolic disease was negative. The IQ followed normal Gaussian distribution. In vitro L-phenylalanine uptake/turnover studies on primary epidermal melanocytes originating from these patients demonstrated a significantly decreased calcium dependent L-phenylalanine uptake and turnover compared to healthy control cells. Based on our observation, we would like to propose that phenylalanine uptake/turnover is under tight control by calcium which in turn could offer an additional novel mechanism in the aetiology of HPA.