Intensive insulin therapy in preschool-aged diabetic children: from multiple daily injections to continuous subcutaneous insulin infusion through indwelling catheters.

In this study, glycemic control, diabetes care indices and quality of life (QoL) were assessed in 2 groups of newly diagnosed Type 1 diabetic subjects <6 yr old who were randomized to multiple daily injections with (Group A) or without (Group B) an indwelling catheter. Group A [12 males (M)/8 females (F), mean age 3.2+/-1.4 yr] and Group B (9M/11F, mean age 3.9+/-1.8 yr) were evaluated at baseline and after 6 and 12 months of treatment. No significant difference was observed in metabolic control (glycosylated hemoglobin) or in the number of hypoglycemic events between the groups. Patients in Group A had a greater number of daily insulin injections, monitored blood glucose more frequently and had a lower total daily insulin dose per kg (p<0.05). QoL was better in group A. At the end of the study 30% of group A patients progressed to continuous sc insulin infusion (CSII), while no child in Group B switched to a different insulin regimen. Based on these findings, indwelling catheter therapy may be helpful for selected CSII candidates.

Age-related differences in metabolic response to continuous subcutaneous insulin infusion in pre-pubertal and pubertal children with Type 1 diabetes mellitus.

The aim of this study was to evaluate clinical and metabolic data in a cohort of Type 1 diabetes (T1DM) children before and after 2 yr of continuous s.c. insulin infusion (CSII). Forty seven T1DM patients were subdivided into two groups: Group A (20 pre-pubertal children, mean age 7.43+/-3.19 yr); Group B (27 pubertal adolescents, mean age 14.47+/-1.91 yr). No statistically significant differences in body mass index (BMI) occurred in either groups after starting CSII or during follow-up. The frequency of mild-hypoglycemias significantly declined during pump therapy only in Group A (p<0.05). Both pre-pubertal and pubertal patients required a significant reduction in their total insulin requirement after 12 and 24 months of CSII. The total percentage of daily insulin doses delivered as basal rates was similar in both groups and was negatively associated (beta=-2.956, p=0.05) with glycosylated hemoglobin (HbA1c) values. No significant correlation was found between the percentage of the basal insulin rate and the number of daily boluses. Differences in timing of the highest insulin requirement were observed between the two groups. Group A had a higher insulin basal rate late in the evening (20:00-24:00 h), while Group B had a higher insulin requirement early in the morning (03:00-07:00 h). The HbA1c levels significantly improved in Group A after 6-12 and 24 months of CSII. In Group B a reduction of HbA1c values was observed only after 6 months of pump therapy (p=0.05). CSII is an effective therapy for all ages but different metabolic requirements should also be taken into account.

Antibody-dependent killing of tumor cells by polymorphonuclear leukocytes. Involvement of oxidative and nonoxidative mechanisms.

Antibody-dependent cellular cytotoxicity (ADCC) against Raji cells was used as a model system to investigate the polymorphonuclear leukocyte (PMN) mechanisms involved in tumor cell lysis. PMN killed target cells by nonoxidative means, as indicated by the following observations: PMN from patients with chronic granulomatous disease (CGD), defective in their metabolic burst, lysed Raji cells normally; impairment of the oxidative metabolism of normal PMN by phenylbutazone did not affect ADCC. Rosenthal's inhibitor of phospholipase A2 completely prevented the lysis, suggesting the involvement of this enzyme in the target cell damage. The inhibition of the Raji cell glutathione redox cycle by carmustine [1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)] increased cell susceptibility to PMN-mediated ADCC. This increment of lysis was related to oxidative killing systems. In fact, CGD PMN had an ADCC against BCNU-treated Raji cells lower than that mediated by normal PMN, but comparable to that observed with untreated targets, and phenylbutazone reduced the lysis of BCNU-treated Raji cells by normal PMN to the level observed with the use of untreated targets. The remaining ADCC against BCNU-treated Raji cells mediated by CGD PMN and by normal PMN in the presence of phenylbutazone was suppressed by Rosenthal's inhibitor. Thus PMN killed sensitized BCNU-treated Raji cells by use of both oxidative and nonoxidative means. The results indicate that the interaction of sensitized Raji cells with PMN triggers simultaneously oxidative and non-oxidative potentially lytic systems and the mediator(s) of Raji cell lysis actually operating may depend on the metabolic state of the target cells themselves. Therefore, the lysis of sensitized tumor cells might not reflect the simple effect of PMN tumoricidal systems; rather it should be regarded as a result of an inability of the target cells to escape the various PMN cytolytic mechanisms.

Serum-associated inhibition of neutrophil Fc receptors in cancer patients.

Rosettes with ox erythrocytes coated with purified IgG antibody were used to detect Fc receptors on neutrophils from 60 patients with solid neoplasias and from 55 normal controls. The mean average of the rosettes in the patients was 48.10%, and that in normal controls was 79.42%, with a highly significant difference according to the Wilcoxon test [negative probability, P(w)-4.47 . 10(-5)]. The low proportion of patients' rosettes ws related to the presence of a serum factor, which also inhibited normal neutrophil rosette formation. Patient neutrophils (or normal neutrophils treated with patient sera) recovered their rosetting capacity when cultured in vitro. No correlation was found between low percentages of rosette-forming cells and the level of circulating immune complexes of the individual patients. Additional evidence also supported the finding that IC and the serum factor are probably unrelated.

Familial aggregation of tumors in the three-year experience of a population-based colorectal cancer registry.

The familial occurrence of tumors has been investigated in 389 subjects with colorectal cancer by reviewing the clinical data and the genealogical tree of all patients registered in 1984-1986, in the Local Health District, for malignancies of the large bowel. Among first-degree relatives of the registered patients there were 89 cases of colorectal cancer as opposed to 19 in a hospital-based control group matched for age and sex [odds ratio (OR), 7.5, P less than 0.001]. This excess of neoplasms among relatives was particularly evident in siblings (60 versus 7, OR 14.7, P less than 0.001) but it was observed also in parents (27 versus 12, OR 4.2, P less than 0.01). Besides colorectal cancer there was no significant excess of other types of tumor in case families, whereas lung tumors tended to be more frequent in control relatives (32 versus 17). Almost half of the registered patients (182 out of 389) had one or more cases of cancer of any sites among relatives; similarly, in 68 there were one or more relatives affected by (or deceased for) colorectal cancer. Moreover, in 27 patients (7.0%) there were at least three cancers of any sites among relatives and in 15 the excess (two or more) was limited to neoplasms of the large bowel. In patients without or with only one neoplasm among relatives, cancers were mainly located in the left colon; however, cancer of the right colon became relatively more frequent in patients with two or more tumors in close relatives. In conclusion, the present study suggests that in approximately 15-20% of patients registered for colorectal cancer one or more first-degree relatives are affected by neoplasms of the large bowel. This familial occurrence of intestinal malignancies (but not of tumors of other organs) strongly suggests a genetic susceptibility to colorectal cancer in a fraction of these patients. Moreover, in a further subgroup of individuals (approximately 5% of all cases) the familial aggregation of two or more cases of colorectal cancer among relatives (besides the proband) and the frequent location of tumors in the right colon make the diagnosis of Lynch syndrome extremely probable.

Fecal neutral steroids in normal conditions and in patients with polyps or cancer of the large bowel.

There is evidence suggesting that the excretion and conversion of neutral sterols in the human large bowel might be somewhat related to the development of colorectal cancer. Therefore, our objectives were: to characterize the excretion and the major pattern of sterol degradation in normal conditions, both in children and in adults; and to investigate if abnormalities of these parameters are frequent in patients with colorectal cancer or polyps. The study has been carried out in: 38 adult volunteers; 29 children divided into 4 age groups; 22 patients with colorectal cancer; 16 members of 6 families with adenomatosis coli; 15 members of 2 families with a high prevalence of multiple polyps or cancer of the large bowel; 12 subjects with colorectal polyps without familiality. With the subjects kept under metabolic control, fecal samples were collected for at least 3 days and analyzed by thin layer chromatography and gas-liquid chromatography. Total neutral steroid excretion was lower in children than in adult volunteers; in contrast, there was no significant difference between the latter and the other investigated group of patients with cancer or polyps, with values ranging between 230 and 680 mg/day. All the adult volunteers were "high converters" of cholesterol to its intestinal metabolites coprostanol and coprostanone [89 +/- 10% (SE) of degradation]. Children less than 1 year old degraded little or no cholesterol (10.4 +/- 6% of total neutral sterols), whereas with increasing age the fraction of conversion became more similar to that of adults. In patients with colorectal tumors 2 populations could be defined, one characterized by a large degradation of cholesterol and the other by little or no conversion. Low degradation of cholesterol was found in 3 of 6 families with adenomatosis coli. In conclusion, we did not find any significant difference in total neutral sterol excretion among controls, colorectal cancer patients, or subjects at risk. In adult volunteers the normal pattern of cholesterol degradation is characterized by a large conversion of cholesterol to its intestinal metabolites. In children this process changes with increasing age from an absolute "nonconverter" state (after birth) to the pattern typical of adults. Finally, in a minority of patients with either polyps or cancer of the large bowel and of their first-degree relatives, cholesterol is poorly degraded and represents the most abundant fecal sterol.

Effects of irradiation and storage on granulocytes harvested by continuous-flow centrifugation.

Five normal subjects were subjected to leukapheresis by continuous-flow-centrifugation (CFC) in the Aminco Celltrifuge. Granulocyte functional capacities were evaluated on the venous blood samples drawn before apheresis and on the cell-rich plasma collected by CFC, immediately after collection and after short-term storage at 4 degrees C with or without previous irradiation (1500 rad, 50 rad/min). The CFC technique has been shown to provide cells without functional damage. Irradiation did not appear to influence granulocyte function, as evaluated by in vitro studies. The data demonstrate that granulocytes maintain, even after irradiation, functional activities similar to those found immediately after collection for up to 24 hours of storage at 4 degrees C and exhibit only a moderate loss of function after 48 h. Chemotaxis appears to be the most sensitive detector of cellular damage of stored granulocytes, either irradiated or non-irradiated; this technique may be the most useful for assessment of granulocyte function before transfusion.

Extracellular cytotoxicity by phagocytosing polymorphonuclear neutrophilic leukocytes: enhancement by a chemotactic stimulus.

This study investigated the influence of a chemotactic stimulus on the extracellular cytotoxicity mediated by phagocytosing polymorphonuclear neutrophilic leukocytes (PMN). We used N-formyl-methionyl-leucyl-phenylalanine (FMLP) as chemotactic peptide, opsonized zymosan as phagocytosable particle, and ox red blood cells (ORBC) as extracellular bystander targets. Phagocytosing PMN were found to kill ORBC efficiently, as determined by the 51Cr-release assay. FMLP, at the concentration of 100 nM, significantly enhanced the target cell lysis. PMN from two patients with chronic granulomatous disease and normal PMN plus catalase or free radical scavengers (mannitol, benzoate, histidine) were completely devoid of cytolytic activity both in the presence and in the absence of FMLP. The results indicate that the target cell lysis by phagocytosing PMN as well as the chemotactic peptide-related amplification of the lysis itself depend on the expression of the PMN oxidative cytotoxic potential. A similar response to a chemotactic stimulus in vivo could provide a mechanism for regulating PMN-dependent cytotoxic and inflammatory processes.

Prostaglandin E2 inhibits apoptosis in human neutrophilic polymorphonuclear leukocytes: role of intracellular cyclic AMP levels.

Human neutrophilic polymorphonuclear leukocytes (neutrophils) are terminally differentiated cells that die by undergoing apoptosis. At present, the intracellular pathways governing this process are only partially known. In particular, although the adenylate cyclase-dependent generation of cyclic AMP (cAMP) has been implicated in the triggering of apoptosis in lymphoid cells, the role of the intracellular cAMP pathway in neutrophil apoptosis remains controversial. In the present study, we found that two cAMP-elevating agents, prostaglandin E2 (PGE2) and the phosphodiesterase type IV inhibitor RO 20-1724, inhibit neutrophil apoptosis without inducing cell necrosis. When administered in combination, PGE2 and RO 20-1724 displayed additive effects. Moreover, neutrophil apoptosis was inhibited by a membrane-permeable analog of cAMP, dibutyryl-cAMP, in a dose-dependent manner. Finally, treatment of neutrophils with the protein kinase A inhibitor H-89 prevented PGE2- and RO 20-1724-induced inhibition of cell apoptosis. In conclusion, taking into account that PGE2 and other cAMP-elevating agents are well known downregulators of neutrophil functions, our results suggest that conditions favoring a state of functional rest, such as intracellular cAMP elevation, prolong the life span of neutrophils by delaying apoptosis.

Autonomic function and autoantibodies to autonomic nervous structures, glutamic acid decarboxylase and islet tyrosine phosphatase in adolescent patients with IDDM.

Recent studies have linked autoimmunity to nervous tissue structures and diabetic autonomic neuropathy, but data on the early stage of IDDM and on the natural history of this association are not available. For this reason, we investigated autonomic nervous function, and the presence of autoantibodies to sympathetic and parasympathetic nervous structures, to glutamic acid decarboxylase (GAD) and tyrosine phosphatase (IA-2/ICA512) in 85 adolescents with insulin-dependent diabetes mellitus (IDDM) (mean age 14.7+/-1.6 yr, mean duration of diabetes 6.8+/-3.5 yr), and 45 age and sex-matched healthy subjects. Nervous tissues autoantibodies were detected using an indirect immunofluorescent complement-fixation technique, with monkey adrenal gland, rabbit cervical ganglia and vagus nerve as substrates. GAD and IA-2/ICA512 autoantibodies were detected by radioimmunoprecipitation assay. Seven patients (8%) had anti-vagus nerve autoantibodies, 7 other patients (8%) had anti-cervical ganglia autoantibodies, while all controls were negative (P < 0.05). Anti-adrenal medulla antibodies were detected in 16 patients (19%) and in 2 control subjects (P<0.02). None of the patients had autonomic symptoms. When patients were divided according to the presence or absence of autoantibodies, values of the cardiovascular tests (deep breathing, 30:15 ratio, Valsalva ratio) were similar in the two groups and similar to those in healthy subjects. However, when considered together, patients positive for one or more autoantibody showed a trend for lower values of deep breathing test and 30:15 ratio test, compared with healthy control subjects, which failed to reach conventional significance values (P=0.17 and P=0.07, respectively). No correlation was found between cardiovascular parameters and metabolic control or diabetes duration. There was no association between autoimmunity to nervous tissue structures and presence of GAD and IA-2/ICA512 Ab, and no correlation between these two autoantibodies and values of cardiovascular tests. Our data indicate that autonomic dysfunction is not a characteristic of young diabetic patients, but that autoantibodies against autonomic nervous structures are present during the first 1 to 15 yr of diabetes. GAD and tyrosine phosphatase appear to be excluded as target autoantigens within autonomic structures. Follow-up studies are required to evaluate future autonomic dysfunction and symptoms in these patients, and to establish whether the subtle autonomic dysfunction detected and/or the nervous tissue autoantibodies, are predictive of the development of this complication.

Cytoprotection against neutrophil-delivered oxidant attack by antibiotics.

In the present study we have investigated the effect of six antibiotics (penicillin G, ceftazidime, cephotaxime, cephoperazon, ampicillin and piperacillin) on the neutrophil cytolytic activity by using a system constituted of phorbol-12-myristate-13-acetate-triggered neutrophils and 51Cr-labelled lymphoblastoid Daudi target cells. The results demonstrate that five of these drugs (ceftazidime, cephotaxime, cephoperazon, ampicillin and piperacillin) are capable of inhibiting the neutrophil cytolytic activity by inactivating the hypochlorous acid (HOCl) generated extracellularly by the myeloperoxidase pathway and crucial to the target cell lysis. Penicillin G had no effect on neutrophil-mediated cytolysis. Thus, these data demonstrate that ceftazidime, cephotaxime, cephoperazon, ampicillin and piperacillin lower the neutrophil-mediated target cell damage by a HOCl-scavenging mechanism, suggesting a possible cytoprotective role for these drugs during infections.

Defective neutrophil mobilization to skin chambers in cancer patients.

The in vivo migration of neutrophils was evaluated in patients affected by epithelial carcinoma using the quantitative skin-chamber technique. The results demonstrated a significant impairment of the patients' neutrophil migration, which was reduced to approximately 4% of that of the controls. Patients' sera were able to inhibit the chemotactic responsiveness of normal neutrophils in vitro. It is therefore suggested that the defective in vivo migration of neutrophils in cancer patients is related to the presence of humoral cell-directed inhibitory activity. This defect of neutrophil function might contribute to the host-defense impairment of carcinoma patients.

Glutamic acid decarboxylase and ICA512/IA-2 autoantibodies as disease markers and relationship to residual beta-cell function and glycemic control in young type 1 diabetic patients.

Circulating autoantibodies (Ab) to islet autoantigens, glutamic acid decarboxylase (GAD(65)), and tyrosine phosphatase ICA512/IA-2 have been proposed as predictive markers of type 1 diabetes mellitus. To ascertain residual beta-cell function and the clinical relevance for monitoring autoimmunity after clinical manifestation of disease, we studied 63 children at diagnosis of type 1 diabetes (mean SD age 7.5 +/- 4 years) and 91 adolescent patients with type 1 diabetes (age 14.7 +/- 1.6 years) with a mean duration of disease of 7 +/- 3.5) years. Forty-two normal adolescent subjects (age 14.6 +/- 1.8 years) without a family history of diabetes were the control group. Anti-GAD(65) and ICA512/IA-2 Ab were assessed by a quantitative radioimmunoprecipitation assay. The relationship between humoral autoimmunity and clinical parameters was explored. GAD(65) and ICA512/IA-2 Ab were detected in 56% and 63% of newly diagnosed children and the prevalence was not different in relationship to clinical characteristics. Levels of GAD(65) Ab positively correlated with diagnosis age (P <.05). Both Ab were associated with islet cell antibodies (ICA) (P <.05), but one fifth of patients had at least 1 of the 2 Ab and absent ICA. At onset, only age showed a significant relationship to residual C-peptide secretion. Among the cohort of patients with diabetes of short-mid duration, GAD(65) and ICA512/IA-2 Ab were present in 44% and 45% of cases (P >.05 and P <.05 v newly diagnosed children, respectively) and more patients were identified by these Ab (68%) than by ICA alone (34%) (P <.05). In this cohort, levels of ICA512/IA-2 Ab negatively correlated with levels of glycosylated hemoglobin (HbA(1c)) (P <.005) and with daily insulin requirement (P <.05). Moreover, the presence of some residual C-peptide secretion was significantly associated with the presence of ICA512/IA-2 Ab (P <.05). Our findings confirm that positivity for either GAD(65) or ICA512/IA-2 Ab is a highly sensitive marker of type 1 diabetes in the pediatric age group, identifying a group of patients with absent ICA immunofluorescence. The persistence of Ab to islet tyrosine phosphatase possibly represents a marker of better glycemic control and less insulin requirement, indicating residual beta-cell function, thus conferring clinical and prognostic relevance to these Ab, as well as potential usefulness in intervention strategies.

Correlation between blood granulocyte progenitor cells and polymorphonuclear leukocytes. A tentative pathophysiological subgrouping of neutropenic and neutrophilic patients.

Blood granulocyte-macrophage progenitors (CFU-GM) were studied in 116 normal, 32 neutropenic and 22 neutrophilic subjects through a double layer agar culture system. The neutropenic group showed significantly lower than normal mean value of CFU-GM per ml of blood, the blood concentration of CFU-GM being within normal limits in 25/32 subjects (78.1%). The neutrophilic group showed significantly higher than normal mean value of blood CFU-GM, and a normal blood concentration of CFU-GM was found in 17/22 patients (77.3%). Within the neutropenic group the concentration of blood CFU-GM was lower than normal in 5/11 (45.4%) patients with less than 1.1 x 10(9) polymorphonuclear leukocytes (PMN) and only in 2/21 (9.5%) patients with more than 1.1 x 10(9)/1 PMN. Within the neutrophilic group the concentration of blood CFU-GM was normal in all 12 subjects having less than 10.5 x 10(9)/1 PMN, while 5/10 (50%) patients with more than 10.5 x 10(9)/1 PMN had higher than normal blood concentration of CFU-GM. The mean leukocyte CSA of the normal, neutropenic and neutrophilic groups did not differ significantly. Within the neutropenic group the CSA was lower than normal in 3/11 (27%) patients with less than 1.1 x 10(9)/1 PMN and in 2/20 (10%) patients with more than 1.1 x 10(9)/1 PMN. Within the neutrophilic group the CSA was normal in all patients with less than 10.5 x 10(9)/1 PMN and it was higher than normal in 2/10 (20%) patients with more than 10.5 x 10(9)/1 PMN. A pathophysiological approach to both neutropenia and neutrophilia, according to PMN and CFU-GM blood concentration, is discussed.

Alterations of erythropoiesis in chronic uremic patients treated with intermittent hemodialysis.

The in vitro growth of the blood burst-forming cells (BFU-E) of 9 chronic uremic patients, treated with intermittent hemodialysis three times a week has been studied at the time of maximum and minimum level of retained nitrogen catabolites. The effect of uremic sera on the vitro growth of normal BFU-E was also studied. The in vitro growth of blood BFU-E was shown to be greatly reduced in all uremic patients and dialysis did not modify their growth. The sera of uremic patients significantly inhibited the in vitro growth of normal blood BFU-E when it was taken at the time of maximum retention of nitrogen catabolites. However, inhibition of normal BFU-E growth was not seen when uremic sera were taken at the time of minimum retention of nitrogen catabolites. These data seem to indicate a long lasting suppression of BFU-E in chronic uremia due to serum inhibitor/s.

Comparison between diurnal changes and changes induced by hydrocortisone and epinephrine in circulating myeloid progenitor cells (CFU-GM) in man.

A significant increase from 8 AM to 3 PM was found in both myeloid progenitor cell (CFU-GM) and polymorphonuclear leukocyte (PMN) blood concentration in 45 normal subjects. Diurnal blood (CFU-GM and PMN changes were significantly correlated. Spontaneous diurnal changes in blood CFU-GM levels and in PMN were compared with the changes induced by i. v. administration of hydrocortisone (16 normal volunteers) and of epinephrine (10 normal volunteers). Diurnal changes in CFU-GM and PMN seem to follow a pattern similar to that induced by epinephrine administration. These findings suggest that diurnal changes in CFU-GM reflect mainly a shift of these cells between different blood compartments.

Evaluation of left ventricular diastolic function in insulin dependent diabetic children by M-mode and Doppler echocardiography.

To evaluate the presence of diabetic cardiomyopathy, we measured various parameters of left ventricular systolic and diastolic function by means of M-mode and Doppler echocardiography in 50 IDDM children (mean age 13 +/- 3 years; mean IDDM duration 5.9 +/- 4.1 years) free of cardiovascular symptoms. As compared to age-matched healthy control subjects, diabetic children evidenced a significant increase in mean values of pressure half time (PHT), an index of the early diastolic phase (53.7 +/- 10.2 msec vs 44.5 +/- 9, p < 0.002). When the patients were subdivided on the basis of IDDM duration, metabolic control and the presence of retinal microangiopathic abnormalities, those with longer IDDM duration and poor glycemic balance had higher PHT values. These data indicate that an early diastolic dysfunction, expressed by reduced left ventricular compliance, can be found in children with Type 1 diabetes mellitus of relatively short duration. Doppler echocardiography is a reliable non-invasive means to assess early impairment of cardiac function in IDDM patients.

Insulin receptors in children's erythrocytes. Study on aging cells.

Aim of this study was to evaluate the influence of the red cell aging process on insulin receptor binding. In erythrocytes from 8 children with high reticulocyte count and 9 healthy age-matched subjects we studied insulin receptor binding in correlation with pyruvate kinase (PK) activity and creatine levels. Moreover maximum 125I-insulin bound % and PK activity were tested in four red cell fractions of different age. Our data show a significant correlation between 125I-insulin bound and either PK activity or creatine levels. In vitro experiments on red cell fractions of different age (as tested by creatine levels) evidence a lower decay of PK activity than insulin bound (p less than 0.05). Our results indicate that creatine content is the best marker of red cell age for insulin receptor studies.

The potential for errors in children with special health care needs.

Children with special health care needs (CSHCN) are at risk for suboptimal treatment when presenting for emergent care to unfamiliar health care providers. Errors in their management may stem from failure to recognize occult conditions, lack of familiarity with rare or complex medical problems, or lack of prior knowledge of baseline physical findings. An emergency information form (EIF) that contains patient-specific information on essential diagnostic and therapeutic interventions may provide a ready personal reference for the emergent care of CSHCN. Coupled with the use of medical identification jewelry and an electronic transmission system, an EIF has the potential to eliminate management errors in the care of these patients.

Tumor cell lysis by activated human neutrophils: analysis of neutrophil-delivered oxidative attack and role of leukocyte function-associated antigen 1.

The lysis of tumor cells, and other nucleated mammalian cells, by neutrophilic polymorphonuclear leukocytes (PMNs) triggered by phorbol myristate acetate (PMA) represents a widely used model system to dissect the PMN cytolytic armamentarium, potentially responsible for the cell damage at tissue sites of PMN activation. Although oxidants are generally considered to be instrumental in the target lysis by PMNs, the mediators actually involved remain a matter of controversy. Moreover, other factors potentially crucial to the lysis have not been clearly identified. In order to reexamine the determinants of the cytolytic process, we studied the events underlying the PMA-triggered PMN-delivered attack against two different targets, selected on the basis of preliminary experiments (B lymphoblastoid Daudi cells and erythroleukemic K 562 cells). The results suggest that the lysis is promoted by hypochlorous acid (HOCl) or a compound with characteristics very similar to HOCl itself. No evidence was obtained for the intervention or contribution of hydrogen peroxide (H2O2), hydroxyl (OH.) radicals, and the major HOCl-derived chloramines. PMNs appeared to use 35% of the generated H2O2 to produce HOCl, while the remainder appears to be consumed by PMNs themselves and target cells as well. Moreover, PMNs and target cells coaggregated at an early step of the cytolytic reaction, through a process efficiently prevented by a monoclonal antibody (MoAb J-90) directed against leukocyte function-associated antigen-1 (LFA-1). The inhibition of the PMN-target aggregation by the MoAb J-90 resulted in the impairment of the lysis, despite a normal generation of HOCl. Thus, the data demonstrate that the PMA-triggered lysis of tumor target cells by PMNs requires at least two events, occurring simultaneously: the LFA-1-mediated effector-target adherence and the PMN production of HOCl. The intervention of the LFA-1-mediated PMN-target adherence in the PMA-triggered lysis is likely to allow PMNs to focus HOCl on the target cell surface and suggests that the process requires a sort of molecule to molecule recognition at the effector-target surface level.