Adil I. Khan

Leukocyte Migration Is Regulated by L-Selectin Endoproteolytic Release

L-selectin mediates lymphocyte migration to peripheral lymph nodes and leukocyte rolling on vascular endothelium during inflammation. One unique feature that distinguishes L-selectin from other adhesion molecules is that it is rapidly cleaved from the cell surface after cellular activation. The biological significance of L-selectin endoproteolytic release was determined by generating gene-targeted mice expressing a modified receptor that was not cleaved from the cell surface. Blocking L-selectin cleavage on antigen-stimulated lymphocytes allowed their continued migration to peripheral lymph nodes and inhibited their short-term redirection to the spleen. Blocking homeostatic L-selectin cleavage also resulted in a constitutive 2-fold increase in overall L-selectin expression by leukocytes. As a result, neutrophils entered the inflamed peritoneum in greater numbers or for a longer duration. Thus, endoproteolytic cleavage regulates both homeostatic and activation-induced changes in cell surface L-selectin density, which directs the migration patterns of activated lymphocytes and neutrophils in vivo.

Role of CD44 and Hyaluronan in Neutrophil Recruitment

Lymphocyte CD44 interactions with hyaluronan localized on the endothelium have been demonstrated to mediate rolling and regulate lymphocyte entry into sites of chronic inflammation. Because neutrophils also express CD44, we investigated the role of CD44 and hyaluronan in the multistep process of neutrophil recruitment. CD44−/− and wild-type control mice were intrascrotally injected with the neutrophil-activating chemokine, MIP-2, and leukocyte kinetics in the cremasteric microcirculation were investigated 4 h subsequently using intravital microscopy. Neither the rolling flux nor the rolling velocities were decreased in CD44−/− mice relative to wild-type mice. In vitro, neutrophils did not roll on the CD44 ligand hyaluronan, consistent with the in vivo data that CD44/hyaluronan did not mediate rolling. However, the number of adherent leukocytes in the venule was decreased by 65% in CD44−/− mice compared with wild-type mice. Leukocyte emigration was also greatly decreased in the CD44−/− mice. The same decrease in adhesion and emigration was observed in the wild-type mice given hyaluronidase. Histology revealed neutrophils as being the dominant infiltrating population. We generated chimeric mice that express CD44 either on their leukocytes or on their endothelium and found that CD44 on both the endothelium and neutrophils was important for optimal leukocyte recruitment into tissues. Of those neutrophils that emigrated in wild-type and CD44−/− mice, there was no impairment in migration through the interstitium. This study suggests that CD44 can mediate some neutrophil adhesion and emigration, but does not appear to affect subsequent migration within tissues.

The variability of results between point-of-care testing glucose meters and the central laboratory analyzer.

CONTEXT Point-of-care testing glucose meters are strongly recommended in the management of diabetes and are increasingly being used for making therapeutically important decisions. Thus, it is essential that their results correlate well with those of laboratory analyzers. OBJECTIVES To test the reliability of point-of-care testing glucose meters. DESIGN Two studies were performed: (1), an in-house study comparing accuracy of point-of-care testing glucose meters with a reference analyzer using fresh whole blood specimens (2), a real-time comparison of (a) 2 successive glucose meter readings and (b) glucose meter reading to central laboratory analyzer reading. SETTING (1), Seven glucose meters from 4 manufacturers were compared with the Yellow Springs YSI 2300 blood glucose analyzer using whole blood without preservative. (2), (a) Whole blood samples were read within 5 minutes of each other using Accu-Chek meters and (b) between a glucose meter and a Hitachi laboratory analyzer. RESULTS (1) Within the Accu-Chek group of glucose meters, fresh, preservative-free whole blood samples showed the lowest bias. (2) At the hypoglycemic level, successive glucose meter readings agreed well, but there was considerable disagreement between glucose meter and central laboratory values. Because laboratory analyzers are of proven accuracy, they are used as the reference. In the glucose meter-central laboratory analyzer correlation, for both hypoglycemic and hyperglycemic values, readings in which the differences were greater than 10% occurred more than 61% of the time. In the hypoglycemic range, differences greater than 20% occurred 57% of the time. CONCLUSIONS One should scrutinize point-of-care testing glucose meter readings at the hypoglycemic and hyperglycemic levels and whenever possible to corroborate these clinical results with central laboratory analyzers.

Procalcitonin: Uses in the Clinical Laboratory for the Diagnosis of Sepsis

Sepsis is the systemic response to infection by microbial organisms. A differential diagnosis of infection caused by either bacteria or other microbial organisms is essential for effective treatment and prognostic assessment. Current clinical laboratory methods in the diagnosis of bacterial infections are either non-specific or require longer turnaround times. Procalcitonin (PCT) is a biomarker that exhibits greater specificity than other proinflammatory markers (eg, cytokines) in identifying patients with sepsis and can be used in the diagnosis of bacterial infections. In this article, we review the current knowledge of PCT and its use in the clinical laboratory setting.

Antiviral activity of natural and semi-synthetic chromone alkaloids

The activity against human immunodeficiency virus (HIV) and herpes simplex virus (HSV), of the non-polar fraction of a methanolic extract of the rootbark of Schumanniophyton magnificum was found to be present in a fraction containing the chromone secondary amine schumannificine 1. Other chromone alkaloids present in the plant were isolated and tested for inhibition of HIV and HSV infections in C8166 and Vero cells, respectively. Acyl and methyl derivatives were prepared and tested. Of all the compounds tested, schumannificine 1 displayed the greatest activity against HIV, whereas potent anti-HSV activity was observed for a number of its derivatives. The presence of a piperidine ring and unsubstituted hydroxy groups on the molecules seems to favour the anti-HIV activity. The anti-HIV activity is considered to be due to irreversible binding to gp120 rather than inhibition of reverse transcriptase or protease.

In Vivo Impairment of Neutrophil Recruitment during Lentivirus Infection

Evidence indicates that the lentivirus, HIV, infection affects neutrophil response to bacteria and bacterial products in vitro. We used a novel model of rapid onset immunosuppression following infection with a similar lentivirus, feline immunodeficiency virus (FIV), in cats to examine neutrophil function within the microvasculature in vivo and to determine the steps that are impaired in the neutrophil recruitment cascade. In uninfected cats and cats infected neonatally with FIV, the mesentery was exteriorized, but remained autoperfused during intravital microscopy for 4 h. When the tissue was superfused with 10 μg/ml of LPS for 4 h, intravital microscopy displayed a profound increase in neutrophil rolling at both 8 and 12 wk of age in uninfected cats. At 12 wk of age, FIV-infected animals showed a profound decrease in the number of rolling neutrophils. In vitro studies revealed that neutrophils from infected and uninfected animals rolled equally well on surrogate selectin substrata. In addition, in vivo neutrophil adhesion and emigration out of the vasculature were severely reduced, and in vitro neutrophil chemotaxis from FIV-infected animals was significantly impaired in response to fMLP or IL-8. However, FIV infection of neutrophils could not be detected. In summary, in vivo lentivirus infection with immunosuppression leads to a severe impairment in neutrophil rolling, adhesion, and emigration in response to bacterial stimulants potentially involving both endothelial and neutrophil dysfunction. These in vivo studies also indicate that neutrophil dysfunction should be taken into account when treating infections and tissue injury.

Lipopolysaccharide: A p38 MAPK-Dependent Disrupter of Neutrophil Chemotaxis

In sepsis, and in models of sepsis including endotoxemia, impaired neutrophil recruitment and chemotaxis have been reported. The inability of the endotoxemic neutrophil to chemotax could be attributed to the fact that intracellular signaling via LPS overrides signals from endogenous chemokines or, alternatively, that sequestration of neutrophils into lungs prevents access to peripheral tissues. Using both in vitro and in vivo chemotaxis assays the authors established that neutrophils from healthy mice chemotaxed in vivo toward MIP‐2, whereas endotoxemic neutrophils did not. Since LPS activates leukocytes via the p38 MAPK pathway, SKF86002, a p38 MAPK inhibitor, was given to endotoxemic animals. SKF86002 significantly reversed the LPS‐induced impairment in emigration of endotoxic neutrophils in response to MIP‐2. Neutrophil chemotaxis in vitro was also impaired by LPS, via a p38 MAPK‐dependent pathway, and this impairment could be reversed via p38 MAPK inhibition. Although neutrophil numbers dropped in the circulation and trapped in lungs during endotoxemia, SKF86002 did not reverse these parameters, demonstrating that p38 MAPK inhibition did not release trapped neutrophils from the lungs. In conclusion, the data suggest that the impaired emigration and chemotaxis of neutrophils at peripheral sites during endotoxemia may be partially due to a p38 MAPK‐mediated inhibition of neutrophil responses to endogenous chemokines.

Synthesis and HIV-1 inhibition of novel benzimidazole derivatives

A range of novel benzimidazole derivatives, some bearing analogy to TIBO, have been synthesized, and evaluated for inhibition of HIV-1 infectivity. The most active and selective compounds are a series of N-alkoxy-2-alkyl-benzimidazoles, several having EC50 < 10μM (one sub-micromolar at 600nM), and selectivity ratios of 10–167. The most selective benzimidazoles, 18a, 18c, show modest RT inhibition, and binding assays indicate gp 120-binding is not a target.

L‐Selectin: An Emerging Player in Chemokine Function

The emigration of leukocytes across the blood‐endothelium barrier and their subsequent transmigration through the interstitium is a complex process that is vital for maintaining the efficiency of the bodys innate and adaptive immunity. The chemokines, a family of low‐molecular‐weight chemoattractant cytokines, are well recognized to be key players in this process. However, recent investigations have highlighted an important role played by the selectin family of adhesion molecules in enhancing chemokine functions. This review summarizes the in vitro and in vivo studies that support this growing notion. It discusses chemotaxis in the context of the phosphoinositide 3‐kinase and p38 mitogen‐activated protein kinase pathways, and their relation to several chemoattractants (i.e., interleukin‐8, leukotriene‐B4, formyl‐methionyl‐leucyl‐phenylalanine, keratinocyte‐derived cytokine, and macrophage inflammatory protein‐2), the possible role played by L‐selectin, and finally how chemotaxis can be altered in different inflammatory settings, such as lipopolysaccharide‐mediated endotoxemia or chronic vasculitis.

Update on Point-of-Surgery Testing

In the approximately 10-year period since the introduction of intra-operative parathyroid hormone testing, a variety of hormones have proven to be valuable tools in the surgical management of patients with various types of resectable tumors. To what extent the current list of hormones/markers performed intraoperatively will expand will be related, in part, to the speed with which new hormones/tumor markers are identified that meet the criteria indicated below for useful markers in point-of-surgery testing (POST). With the exception of intra-operative parathyroid hormone (ioPTH) testing, testing for the other hormones indicated below is performed at specialized centers using either modified commercial (“home-brew”) or research use only (RUO) assays.