Assessing the delivery of neutrophils to tissues in neutropenia

Studies of neutrophil kinetics in neutropenic individuals, as well as clinical observations of variability in the occurrence of infection among patients with neutropenia, have suggested that blood neutrophil counts may not uniformly reflect the effective delivery of neutrophils to extravascular tissues where the cells perform their principal host defense functions. To evaluate this possibility we developed a sensitive, reproducible method of measuring the extravascular delivery of neutrophils to a normal mucosal site of neutrophil turnover. This method is based upon the quantification of neutrophils recoverable from saline mouth wash specimens. Twenty-five mL specimens, obtained in a controlled manner from neutropenic patients and normal subjects, were centrifuged and the sediments resuspended in 1.0 mL Hank's buffer with 2 micrograms acridine orange, incubated at 37 degrees C for 15 minutes, and then examined in a hemocytometer chamber by fluorescence microscopy. Neutrophils could be clearly distinguished by their characteristic fluorescence and were counted. With this method as few as 1,500 neutrophils were detected reliably in mouth wash specimens. Mucosal neutrophil counts varied less than 10% with repeated sampling of individual subjects over 5-day periods and were consistently greater than 1.3 X 10(5)/specimen in non-neutropenic individuals. Although profound neutropenia was generally reflected by lower than normal oral mucosal neutrophil counts, these counts were significantly higher in individuals with chronic severe neutropenia (blood neutrophils less than 300/mm3) than in patients with acute neutropenia of comparable severity that had developed following chemotherapy. Also, in individuals recovering from profound neutropenia, neutrophils usually reappeared earlier in mouth wash specimens than in blood, and oral mucosal neutrophil counts attained recovery levels more rapidly than did blood counts. This phenomenon was particularly evident in an individual with cyclic neutropenia. Moreover, mucosal neutrophils could occasionally be detected in profoundly neutropenic patients when neutrophils were not present in blood samples. These findings indicate that mucosal neutrophil counts in individuals with neutropenia provide information about the delivery of neutrophils to tissues that may not be apparent from blood neutrophil counts alone.     

Excessive apoptosis of bone marrow erythroblasts in a patient with autoimmune haemolytic anaemia with reticulocytopenia

We report a patient with autoimmune haemolytic anaemia (AIHA) with reticulocytopenia, who showed excessive apoptosis of erythroblasts. Ultrastructural analysis of bone marrow cells showed that 50% of erythroblasts had characteristic features of apoptosis, which was confirmed by staining with Annexin-V. In addition, in contrast to normal erythroblasts, Ig staining of the entire erythroblast population could be shown. These data show that apoptosis may contribute to the mechanism of reticulocytopenia in AIHA.     

Methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii: An unexpected difference in epidemiologic behavior

Background: The Dutch guideline on hospital policy for the prevention of nosocomial spread of methicillin-resistant Staphylococcus aureus (MRSA) states that patients transferred from hospitals abroad must be placed in strict isolation immediately on admission to a hospital in the Netherlands. Three patients colonized with both MRSA and a multiresistant Acinetobacter were transferred from hospitals in Mediterranean countries to 3 different hospitals in the Netherlands. Despite isolation precautions, Acinetobacter spread in 2 of the 3 hospitals, whereas nosocomial spread of MRSA did not occur. Methods: For outbreak analysis, the Acinetobacter isolates, identified as Acinetobacter baumannii by the use of amplified ribosomal DNA restriction analysis, were comparatively typed by 4 methods. Comparison of isolation measures in the hospitals was performed retrospectively. Results: In the 2 hospitals in which nosocomial spread of Acinetobacter occurred, most of the epidemiologically related isolates were indistinguishable from the index strains. In these 2 hospitals, isolation measures were in concordance with those recommended for the prevention of contact transmission. The precautions of the hospital in which no outbreak occurred included the prevention of airborne transmission. Conclusions: Precautions recommended for multiresistant gram-negative organisms are insufficient for the prevention of nosocomial spread of multiresistant Acinetobacter . The airborne mode of spread of acinetobacters should be taken into account, and guidelines should be revised accordingly. (AJIC Am J Infect Control 1998;26:544-51)     

Group 2 innate lymphoid cells in lung inflammation

Although allergic asthma is a heterogeneous disease, allergen‐specific T helper 2 (Th2) cells producing the key cytokines involved in type 2 inflammation, interleukin‐4 (IL‐4), IL‐5 and IL‐13, are thought to play a major role in asthma pathogenesis. This model is challenged by the recent discovery of group 2 innate lymphoid cells (ILC2) that represent a critical innate source of type 2 cytokines. These ILC2 are activated by epithelial cell‐derived cytokines, including IL‐25 and IL‐33, which have been implicated in the initiation of asthma. In this review, we will discuss recent studies supporting a significant role for ILC2 in lung inflammation, with special attention to allergen‐induced asthma.     

Amplification of genes encoding human myeloid membrane antigens after DNA-mediated gene transfer

Spontaneous amplification of genes encoding two different human myeloid surface antigens was observed after DNA-mediated gene transfer of cellular DNA from the human myeloid cell line HL-60 into NIH-3T3 mouse fibroblasts. Transformed recipient cells with highly amplified expression of either of two donor membrane polypeptides, gp150 or p67, were isolated with a fluorescence-activated cell sorter (FACS), using monoclonal antibodies specific for human myeloid cells. Immunoprecipitation of enzymatically radioiodinated polypeptides from the surface of transformed NIH-3T3 cells confirmed that expression of these proteins was amplified tenfold to 20-fold in comparison to their expression on human myeloid cell lines. The cellular DNA of cloned secondary and tertiary transformants expressing high levels of gp150 and p67 contained amplified sets of DNA restriction fragments that hybridized with human repetitive DNA sequences. Cytogenetic analysis of subclones overexpressing gp150 revealed extrachromosomal double minutes (DMs), whose presence correlated with the unstable expression of the membrane polypeptide. Human sequences in gp150-positive clones did not localize to chromosomes, consistent with their association with extrachromosomal DMs. By contrast, p67-positive subclones stably expressed the antigen, and in situ hybridization to metaphase spreads demonstrated that amplified human DNA sequences were integrated into a specific marker chromosome. Cytogenetic analysis of the parental NIH- 3T3 subclone used in these studies disclosed DMs in a low percentage of metaphases, suggesting that the recipient cells have a propensity for amplifying donor DNA.     

Molecular heterogeneity in acute leukemia lineage switch

Six cases of acute leukemia that underwent lineage switch from acute lymphocytic leukemia to acute myelogenous leukemia are reported. The mean age of the patients was 24 years, time to conversion was 36 months, and survival after conversion was only 3 months. Of the three cases which showed abnormal metaphases at both diagnosis and conversion, two (cases 2, 5) showed related cytogenetic abnormalities, and the third showed (case 3) independent chromosomal changes. Molecular analysis for immunoglobulin heavy chain and T-cell receptor beta chain genes showed that five of the six cases had rearrangement of at least one of these lymphoid associated genes at conversion to acute myelogenous leukemia. The single case (case 3) in which there were no lymphoid gene rearrangements at conversion was also the only case in which independent karyotypic abnormalities at diagnosis and conversion were demonstrated. Our findings suggest that lineage switch can represent either relapse of the original clone with heterogeneity at the molecular level or the emergence of a second new leukemic clone without molecular heterogeneity.     

Postoperative changes in collagen synthesis in intestinal anastomoses of the rat: differences between small and large bowel.

Collagen synthesis is an essential feature of anastomotic healing in the intestine. Postoperative collagen synthesis, measured in vitro in intestinal anastomoses was studied from three hours to 28 days after operation. For this purpose, an ileal and a colonic anastomosis were constructed within the same animal and the results in both intestinal segments were compared. In the ileum, collagen synthesis was significantly increased, with respect to unoperated controls, three hours after operation. It remained raised during the period of study, with a maximal 10-fold stimulation four days after operation, and had nearly returned to the preoperative level after four weeks. The general pattern was the same in the colon, although quantitatively different: the increase in synthetic activity was delayed in comparison with the ileum. Maximal stimulation was approximately six-fold. In addition, we calculated the ratio for each rat between anastomotic collagen synthesis and the average value found in non-operated control animals. Postoperative stimulation in the ileum was higher than in the colon in almost every animal examined. The results show that the ileum responds more quickly and strongly to wounding than the colon, at least as far as the production of new collagen is concerned. Possibly, this phenomenon contributes to the lower failure rate apparent for anastomoses in the small bowel.