G. Santini

Age-related changes in human lymphocyte subsets: Progressive reduction of the CD4 CD45R (suppressor inducer) population

The purpose of this study was to investigate, by double-labeling immunofluorescence, lymphocyte subsets in the peripheral blood of healthy children, adults, and aged individuals. The absolute number of T and B lymphocytes decreases with age. The decline in T cells was attributed to a decrease in CD4 lymphocytes. We also found that the composition of the CD4 subset changes with age: in children the CD45R molecule is expressed on the majority of CD4 cells, whereas in aged subjects the absolute number of these lymphocytes is greatly reduced. The reciprocal CD4 CD29 population is not modified during the life span. Aging is also associated with the appearance of CD8 Leu 7 lymphocytes. Putative contrasuppressor cells, identified by Vicia villosa binding, represent a very small population in peripheral blood and are not subject to age-dependent variations.

A subset of γδ lymphocytes is increased during HIV-1 infection

The γδ T cell receptor (TcR) lymphocytes constitute 3–10% of human peripheral blood lymphocytes. Only a very small fraction of these cells is recognized by the δTCS1 monoclonal antibody, directed against the Vγδ1 chain of the receptor. We describe the immunological, virological and clinical data of a small group of seropositive subjects having high levels of γδ TcR T cells in the peripheral blood. Our flow cytometric studies show that most of these cells belong to the δTCS1+ (Vδ1+), CD8± (dim staining) subset. Patients with high γδ TcR T cell numbers were not characterized by the presence of an acute (IgM positive) or reactivated (as defined by high IgG litres against early antigen or IgA titres against viral capsidic antigen) Epstein‐Barr virus infection. Cytomegalovirus infection was excluded by serological assays, and other herpesviral infections were not found after clinical examination. HIV p24 antigenaemia was present in two out of 11 subjects. AIDS patients had very high percentages of γδ TcR T cells. Altogether these data show that the selective expansion of δTCS1+ cells in HIV1 seropositive subjects is not related to some exogenous antigen stimulation, but may be related to peculiar pathologic processes involving the immune system.

SOURCE AND ROUTE OF MICROBIAL COLONISATION OF PARENTERAL NUTRITION CATHETERS

To assess the effectiveness of tunnelling the polyurethane venous catheter for parenteral nutrition in reducing the frequency of catheter microbial colonisation, and to investigate the routes taken by microorganisms colonising the central venous catheter, 109 patients were randomised to traditional subclavian catheterisation (58, group A) or to subcutaneous catheter tunnelling (51, group B). Samples were taken from patients and their nurse attendants to identify their indigenous flora. Cultures were also done of swabs from the catheter insertion site, blood, nutrient solution, segment of the catheter, and washings of the catheter hub. Intravascular segment colonisation was commoner in group A (18/58) than in group B patients (4/51), and bacterial migration from insertion site to intravascular segment was also commoner among group A (9/58) than among group B patients (1/51). Catheter hub contamination was responsible in 10 out of 22 cases of microbial colonisation; in 6 of these 10 the bacterium isolated was present on the skin of nurses who changed the bag. Contamination of the insertion site skin and of the CVC hub were equally responsible for the microbial colonisation of the intravenous segment of the catheter.

Time-dependent efficacy of bacterial filters and infection risk in long-term epidural catheterization.

BACKGROUND: Epidural infection represents a serious albeit infrequent complication of long-term epidural catheterization. The catheter hub is regarded as the main point of entry for microorganisms among the three possible routes (hematogenous, insertion site, hub) of microbial colonization of the inserted catheter. The current study was aimed at evaluating whether frequent changing of antimicrobial filters carries an increased risk of catheter hub contamination and the time-dependent efficacy of commonly used antimicrobial filters after prolonged use. METHODS: In the first part of the study, a microbiologic survey (skin, filter, hub, and catheter tip) was performed weekly in a group of 47 patients with cancer bearing subcutaneously tunneled catheters managed at home. Subsequently, the time-dependent efficacy of 96 micropore filters (32 Portex, 32 Sterifix-Braun, 32 Encapsulon TFX-Medical) differing in surface areas and/or composition of the filtering membrane was evaluated in a laboratory study. Filters were perfused, under the usual conditions of clinical use (flow resistance, injection pressure, temperature), every 8 h up to 60 days, with 5 ml of two different analgesic solutions, either sterile or containing 1.5 x 10(5)/ml of Streptococcus milleri I. Eight filters of each type subsequently were flushed with a S. milleri suspension (0.5 McFarland) after 7, 14, 28, and 60 days of continuous perfusion, and the resulting filtrates were cultured. RESULTS: In 16 of 19 positive hub cultures, the same microorganisms (species, biotype, antibiotype) were cultured from skin and filters. A statistically significant positive trend was found between the number of filter changes and the rate of positive hub cultures (chi 1(2) trend 5.11; P = 0.02). A high correlation coefficient was found between number of positive skin cultures and number of positive filtrates (r = 0.88; P = 0.01) and between number of positive filtrates and number of positive hub cultures (r = 0.93; P = 0.003). Cultures obtained from Portex and Sterifix-Braun filters yielded no bacterial growth (64/64) throughout the study period. Cultures from Encapsulon TFX-Medical filters showed bacterial growth 2/8 at seventh day, 7/8 at the 14th day, and 16/16 from the 28th day onward. CONCLUSIONS: Our data indicate significant correlation between the incidence of catheter hub colonization and the filter-change frequency, when the skin close to the filter-hub connection is contaminated. Our results also show that Portex and Sterifix-Braun bacterial filters, when perfused with reduced volumes at low injection pressures, maintain an unmodified antimicrobial function for at least 60 days. Based on these data, it appears clinically feasible to reduce the frequency of filter changes during long-term epidural catheterization, with a consequent possible decrease of epidural catheter colonization.

Enumeration of human lymphocyte subsets by monoclonal antibodies and flow cytometry: a comparative study using whole blood or mononuclear cells separated by density gradient centrifugation

Enumeration of lymphocyte subpopulations by combined use of flow cytometry and monoclonal antibodies is influenced by the separation method used. T or B lymphocyte antigen frequencies do not differ in samples of whole blood or after separation on Ficoll-Paque or Percoll, but there is a significant increase of Leu+ Leu11+ lymphocytes showing strong natural killer activity at the sample-separation medium interface. At the bottom of the tubes a selective loss of OKT+, Leu7+, Leu11- cells is found; at this level cytotoxicity is very low. Our data suggest that Leu7+ cells could be subdivided into 2 subpopulations differing in reactivity with the monoclonal antibody to Leu11, natural killer activity and density. Differences observed in the percentages of Leu7+, Leu11+ lymphocytes between whole blood and separated cells could be due to an enrichment produced by centrifugation or to the presence in whole blood of a factor interfering with antibody binding.

A method for the determination of the adherence of granulocytes to microtitre plates

We report a new partially automated method for the measurement of the adherence of PMN in vitro. Adherence to a plastic surface was detected by measuring leukocyte alkaline phosphatase activity of the adherent cells, with a Titertek Multiscan system. Using three different cellular concentrations (1 X 10(6), 5 X 10(5), 2.5 X 10(5) PMN/ml) the response curve was linear to 45 min and adhesion was maximal by 30 min. The specificity of the reaction was acceptable as was the assay reproducibility (intra-assay coefficient of variation less than 8%; inter-assay coefficient of variation less than 11%).

Close genetic linkage between HLA and renal glycosuria

Renal glycosuria is an inherited disorder of renal tubule function in which significant amounts of glucose are excreted in the urine in the simultaneous presence of normal blood glucose levels. Renal glucose titration analyses and HLA genotypes were performed in 5 unrelated affected families with a total of 25 patients and 40 healthy relatives. In each family the gene responsible for renal glycosuria segregates with the HLA complex suggesting a close genetic linkage. 2 cases carry intra-HLA recombinant haplotypes; in these subjects our findings indicate that the abnormal gene is closer to the HLA-A locus than the HLA-B locus. No HLA-A, HLA-B or HLA-C specific antigen is selectively increased among the 5 unrelated families affected with renal glycosuria.

Torulopsis glabrata Peritonitis Complicating Continuous Ambulatory Peritoneal Dialysis: Successful Management With Oral 5-Fluorocytosine

We report two cases of fungal peritonitis caused by Torulopsis glabrata, an uncommon opportunistic pathogen, in patients with end-stage renal disease receiving continuous ambulatory peritoneal dialysis (CAPD). The general clinical characteristic of T glabrata peritonitis was comparable to previously reported cases of Candida peritonitis. Although appropriate therapy of fungal peritonitis in patients undergoing CAPD still remains controversial, both for the drug of choice and for the dosage to be used, our study indicates that a 5-week course of oral 5-fluorocytosine (5-FC) may obviate the need to remove the peritoneal catheter during the management of peritonitis caused by susceptible strains of T glabrata.

Selective depletion of the OKT 4+ 4B4+ subset in lymph nodes from HIV+ patients

We have used 4B4 and 2H4 monoclonal antibodies in conjunction with OKT 4 to quantify T cell subsets in lymph node suspensions from human immunodeficiency virus (HIV) positive subjects with lymphadenopathy syndrome. The data indicate that the reduced OKT 4:OKT 8 ratio was due to a depletion of the OKT 4+ 4B4+ subset. In contrast, there were no differences compared to reactive controls, considering the OKT8+ subpopulation. These alterations may be related to the immunological deficiency associated with HIV infection.

Phenotypic analysis of a CD2− CD3+ T cell receptor gamma delta lymphocyte subset

We have identified, in a patient with atopic dermatitis, a consistent population of peripheral blood lymphocytes expressing a CD3+ gamma/delta TCR complex, while being unreactive with CD2. Further immunofluorescence studies showed that these cells almost completely co-express CD29 and CD45RA and have high membrane levels of CD11a compared to the alpha/beta TCR T cells. Neither a genetic influence nor an acute or reactivated herpesvirus infection were found to be related to the expanded gamma/delta T-cell subpopulation. Our data confirm the previous observations regarding the presence in the peripheral blood of an expanded gamma/delta TCR, CD2- subset and show that these cells have a peculiar phenotypic profile. The reasons for this expansion are, however, still unknown.