Which agonist properties are important for the activation of 5-HT3A receptors?

PURPOSE: Why do anesthetics not activate excitatory ligand-gated ion channels such as 5-HT3 receptors in contrast to inhibitory ligand-gated ion channels? This study examines the actions of structural closely-related 5-HT derivatives and 5-HT constituent parts on 5-HT3A receptors with the aim of finding simpler if not minimal agonists and thus determining requirements for successful agonist action. EXPERIMENTAL APPROACH: Responses to 5-HT derivatives of human 5-HT3A receptors stably expressed in HEK 293 cells have been examined with the patch-clamp technique in the outside-out configuration combined with a fast solution exchange system. RESULTS: Phenol, pyrrole and alkyl amines, constituents of 5-HT, even at high concentrations, cannot activate 5-HT3A receptors but they can inhibit them. To date, tyramines are the smallest known agonists. However, an aromatic ring is not required for activation as acetylcholine is also an agonist of similar strength. CONCLUSION: Simultaneous interactions of adequate strength at two separate subsites within the 5-HT binding domain appear to be essential for successful agonist function. Anesthetics either fail to achieve this or the activation they produce is so weak that it is masked by a comparatively very strong inhibition.

Shifting priorities in the aftermath of a Q fever epidemic in 2007 to 2009 in The Netherlands: from acute to chronic infection.

From 2007 to 2009, the Netherlands faced large seasonal outbreaks of Q fever, in which infected dairy goat farms were identified as the primary sources. Veterinary measures including vaccination of goats and sheep and culling of pregnant animals on infected farms seem to have brought the Q fever problem under control. However, the epidemic is expected to result in more cases of chronic Q fever among risk groups in the coming years. In the most affected area, in the south of the country, more than 12% of the population now have antibodies against Coxiella burnetii. Questions remain about the follow-up of acute Q fever patients, screening of groups at risk for chronic Q fever, screening of donors of blood and tissue, and human vaccination. There is a considerable ongoing research effort as well as enhanced veterinary and human surveillance.

Isolated blunt chest injury leads to transient activation of circulating neutrophils.

INTRODUCTION: The acute respiratory distress syndrome (ARDS) is a severe and frequently seen complication in multi-trauma patients. ARDS is caused by an excessive innate immune response with a clear role for neutrophils. As ARDS is more frequently seen in trauma patients with chest injury, we investigated the influence of chest injury on the systemic neutrophil response and the development of ARDS. MATERIALS AND METHODS: Thirteen patients with isolated blunt chest injury [abbreviated injury score (AIS) 2-5] were included. To avoid systemic inflammation caused by tissue damage outside the thorax, injuries to other regions than the chest did not exceed an AIS of 2. At 3, 9 and 24 h after injury, the expression of circulating activating molecules on neutrophils and levels of circulating interleukine (IL)-6 were determined. Blood samples from eight healthy volunteers were used as control. RESULTS: Blunt chest injury resulted in the activation of circulating neutrophils, as characterized by a decreased expression of l-selectin (CD62L), CXCR2 (CD182b) and C5aR (CD88) compared to control (p < 0.05). Expression of l-selectin, CXCR2 and C5aR was partially restored at 24 h after injury. In addition, the mean expression of FcγRIII (CD16) dropped (p < 0.001), indicating the recruitment of young neutrophils into the circulation. IL-6 levels increased to a maximum mean concentration of 86 ± 31 pg/ml at 24 h postinjury. None of the patients developed ARDS. CONCLUSION: Blunt chest trauma caused a systemic inflammatory reaction with transient activation of neutrophils and mobilization of young neutrophils into the circulation. Isolated chest injury, however, was not abundant enough to cause ARDS, so a second hit appears crucial.

Use of antiviral agents and other measures in an influenza pandemic.

The Dutch Ministry of Health asked the Health Council for advice on how to prepare for a possible influenza pandemic. In two advisory reports the Committee responsible indicated the measures that it believes would need to be taken if such a pandemic were to reach the Netherlands. During a pandemic, the Committee recommends that every resident of the Netherlands with influenza-like illness should be treated with neuraminidase inhibitors such as antiviral agents. This approach serves to mitigate the course of the disease, to reduce infectivity and to allow patients to build up immunity to the virus. Since up to 30% of the population could become ill, the Committee anticipates that a stock of five million courses of the neuraminidase inhibitor oseltamivir is sufficient. If a pandemic were to occur at a time that the stock does not exceed the present 225,000 courses, the committee advises restricting treatment to three specified groups of patients. If the first few patients are traced shortly after they fall ill, the Committee recommends treatment of the patient and postexposure prophylaxis for his/her close contacts. The Committee does not advocate prophylaxis in general, but it can envisage prophylaxis for particular groups of patients or under particular circumstances. The Committee believes that in order to reduce rapid spread of the virus, schools should be closed and events where large numbers of people gather in a confined space should be cancelled. Because this recommendation would have major social and economic consequences, the Committee understands that its implication will depend on the anticipated severity and extent of the pandemic. The Committee regards vaccination against influenza as the best means of protecting the population. The development of a vaccine should be the absolute priority.

Blood products and parvovirus B19.

A Committee of the Health Council of The Netherlands has expressed its opinion on introducing testing of blood products for parvovirus B19 (B19). Although infections with B19 generally run their course without any serious health problems, for some groups, such as pregnant women, patients with underlying haematological problems and patients with immunodeficiency, infections with B19 can result in serious complications. For cellular blood products, which are derived either from a single donor or a limited number of donors and are administered either to a single patient or to a limited number of patients, the Committee recommends that a risk-group approach be adopted and that 'Big-virus safe' blood products be administered to the risk groups mentioned above. The Committee defines as 'Big-virus safe' cellular blood products from a donor in which IgG antibodies against B19 have been detected in two separate blood samples, one taken at least six months after the other. Patients other than those in the risk groups should continue to receive cellular blood products that have been produced in accordance with current safety criteria. For plasma products, which are prepared from plasma pools and are administered to large numbers of patients, the measures must be aimed at cutting down the levels of B19 infectivity in such pools. For plasma pools, the Committee proposes a maximum permissible limit of 104 genome copies of Bl9 per ml.

Induction of clinical remission in T-large granular lymphocyte leukemia with cyclosporin A, monitored by use of immunophenotyping with Vbeta antibodies.

A 54-year-old woman presented with a severe autoimmune anemia, thrombocytopenia, neutropenia (Evans' syndrome), and CD8+ lymphocytosis, without signs of lymphadenopathy or splenomegaly. A diagnosis of T cell large granular lymphocyte (T-LGL) leukemia was made, based on cytomorphology, the typical CD3+/CD4-/CD8+/CD16+/CD56-/CD57-/HLA-DR(+/-) immunophenotype of the lymphocytosis (9 x 10(9)/l), and biallelic clonally rearranged T cell receptor beta (TCR beta) genes. Clonality of the TCR alphabeta+ T-LGL was also demonstrated with a panel of antibodies against variable domains of TCR beta chains, which showed single Vbeta7.1 expression on the CD3+ T-lymphocytes. After treatment failure with corticosteroids, splenectomy, and cyclophosphamide, respectively, a complete clinical remission was induced and sustained with cyclosporin A. Vbeta7.1/CD8/CD3 triple immunofluorescence stainings appeared to be valuable for titrating the cyclosporin A dosage by monitoring the T-LGL cells during treatment.

Immunophenotyping of blood lymphocytes in childhood. Reference values for lymphocyte subpopulations.

OBJECTIVE: Immunophenotyping of blood lymphocytes is an important tool in the diagnosis of hematologic and immunologic disorders. Because of maturation and expansion of the immune system in the first years of life, the relative and the absolute size of lymphocyte subpopulations vary during childhood. Therefore we wished to obtain reference values for the relative and the absolute size of all relevant blood lymphocyte subpopulations in childhood. STUDY DESIGN: We used the lysed whole blood method for analysis of lymphocyte subpopulations in 429 blood samples from neonates (n = 20), healthy children (n = 358), and adults (n = 51). The following age groups were used: 1 week to 2 months (n = 13), 2 to 5 months (n = 46), 5 to 9 months (n = 105), 9 to 15 months (n = 70), 15 to 24 months (n = 33), 2 to 5 years (n = 33), 5 to 10 years (n = 35), and 10 to 16 years (n = 23). RESULTS: Our results show that the absolute number of CD19+ B lymphocytes increases twofold immediately after birth, remains stable until 2 years of age, and subsequently gradually decreases 6.5-fold from 2 years to adult age. The CD3+ T lymphocytes increase 1.5-fold immediately after birth and decrease threefold from 2 years to adult age. The absolute size of the CD3+/CD4+ T-lymphocyte subpopulation follows the same pattern as the total CD3+ population, but the CD3+/CD8+ T lymphocytes remain stable from birth up to 2 years of age, followed by a gradual threefold decrease toward adult levels. In contrast to B and T lymphocytes, the absolute number of natural killer cells decreases almost threefold in the first 2 months of life and remains stable thereafter. Our study also showed that changes in the absolute size of lymphocyte subpopulations are not always consistent with changes in their relative size. This demonstrates that the relative counts of lymphocyte subsets do not reflect their actual size and are therefore of limited value. CONCLUSION: On the basis of this study we strongly recommend that immunophenotyping of blood lymphocytes for the diagnosis of hematologic and immunologic disorders be based on the absolute rather than on the relative size of lymphocyte subpopulations. Our data can be used as age-matched reference values for blood lymphocyte immunophenotyping.

Four aged siblings with B cell chronic lymphocytic leukemia.

All four aged siblings (>80 years) of one family presented with B cell chronic lymphocytic leukemia (B-CLL). In an attempt to find common characteristics in the four patients, we performed detailed immunological marker analysis, Southern blot analysis of immunoglobulin (Ig) genes, and cytogenetic studies. In three patients clonality of the B-cells could be proven by single Ig light chain expression, but in the fourth patient no Ig light chain expression was detected and clonality of the B cells could only be demonstrated by Southern blot analysis of the Ig genes. Interestingly, in two patients, the Ig gene rearrangement patterns were compatible with the presence of two independent B cell clones, whereas in the two other siblings a monoclonal rearrangement pattern was found. All four patients showed clonal chromosome aberrations, which were different in each patient. In the two patients with biclonal Ig gene rearrangement patterns, two unrelated clones could also be demonstrated by the cytogenetic studies. These combined Ig gene and cytogenetic data indicate the presence of two different B-CLL in two of the four patients. Remarkably, the B-CLL cells of the two oldest patients expressed the CD8 antigen, which is rarely observed. Our finding of six different B-CLL in the four living siblings indicates that the members of this family are highly susceptible to the development of B-CLL. However we could not identify a common factor to explain this susceptibility further. In contrast to the literature, the occurrence of two B-CLL in one patient and the expression of CD8 were not associated with clinically aggressive disease in this family.

Flow cytometric detection of intracellular antigens for immunophenotyping of normal and malignant leukocytes.

Intracellular antigens are of major importance for immunophenotyping of normal leukocytes as well as leukemias and malignant lymphomas. Immunofluorescence microscopic evaluation of cytocentrifuge preparations has remained the preferred technique for detection of intracellular antigens for a long time. Recently, flow cytometric detection of intracellular antigens has been improved by the development of new permeabilization/fixation solutions. We compared four commercially available solutions: FACS Brand Lysing Solution (FACS Brand; Becton Dickinson, San Jose, CA, USA), Fix & Perm cell permeabilization kit (Fix & Perm; An der Grub, Vienna, Austria), OptiLyse B lysing solution (OptiLyse B; Immunotech, Marseille, France), and ORTHO PermeaFix(PermeaFix; Ortho Diagnostic Systems, Raritan, NJ, USA). These solutions were evaluated for the complexity and duration of the intracellular staining procedure, the effects on light scatter patterns, and the staining results for the intracellular antigens terminal deoxynucleotidyl transferase (TdT), cytoplasmic CD3 (CyCD3), myeloperoxidase (MPO), and cytoplasmic immunoglobulin light chains (CylgL). The four methods could easily be introduced in our laboratory and had only minor effect on the light scatter patterns of the tested cell samples. Each of the four tested antigens was detectable with at least one of the four methods. Only the Fix & Perm cell permeabilization kit could be used for reliable detection of all four intracellular antigens. In a large series of 450 BM and PB samples containing various percentages of TdT+ cells, the results of flow cytometric TdT staining with FACS Brand Lysing Solution were highly comparable to the results obtained by immunofluorescence microscopy (P = <0.00001). Our comparative study shows that flow cytometric detection of the intracellular antigens TdT, CyCD3, MPO, and CylgL can now reliably be performed on a routine basis.

Detection of minimal residual disease in acute leukemia patients.

Diagnostic techniques, routinely used in clinical practice for monitoring acute leukemia patients, are able to detect only 1-5% of malignant cells. At present, two main techniques are being introduced for detection of minimal residual disease (MRD) in leukemia, namely immunological marker analysis and the polymerase chain reaction (PCR) technique with general sensitivity of 10(-4)-10(-5). Immunological marker analysis allows detection of unusual and aberrant immunophenotypes, and is usually performed by flow cytometry. PCR analysis allows detection of leukemia-specific DNA sequences, such as fusion regions of chromosome aberrations and junctional regions of rearranged immunoglogulin (Ig) genes and T-cell receptor (TcR) genes. The applicability of the immunophenotyping and PCR-mediated MRD techniques is dependent on the type of leukemia. In virtually all acute lymphoblastic leukemias, PCR analysis of Ig and TcR genes can be used, and immunophenotypic MRD detection is also possible in 70-80% of cases. In AML, immunophenotypic MRD detection can be applied in approximately 80% of cases and PCR analysis of chromosome aberrations in 25-40%. Each MRD technique has its advantages and limitations, which have to be weighed carefully to make an appropriate choice. Furthermore, standardization of the MRD techniques is needed before they are used for stratification or adaptation of treatment protocols. Finally, the clinical impact of MRD detection for the various subtypes of acute leukemias has to be established.

Cytogenetic clonality analysis in myelodysplastic syndrome: monosomy 7 can be demonstrated in the myeloid and in the lymphoid lineage.

Bone marrow and blood from three patients with myelodysplastic syndrome (MDS) and monosomy 7 were studied for cell lineage involvement of the chromosomal abnormality. Cytogenetic involvement of the myeloid and erythroid cell lineages in MDS with monosomy 7 has been shown before. Lymphoid subpopulations have also been investigated but generally with negative results. A combined technique of May-Grünwald-Giemsa (MGG) for cell cytology and interphase fluorescence in situ hybridization (FISH) using a chromosome 7 specific DNA probe was applied. Further, immunophenotype and genotype of the cells were simultaneously examined with alkaline phosphatase anti-alkaline phosphatase (APAAP) immunostaining and FISH. The monosomy 7 was found in the blasts and in all or in subpopulations of myeloid and erythroid cells. T cells (CD3+, CD5+) did not appear to be involved. B cells (CD19+, CD22+) showed a normal distribution of FISH spots in two patients. In one patient however the loss of a chromosome 7 was found in approximately 70% of the cells positive for B cell markers including CD79a. The results of this study show that in some cases MDS is a disease arising in a progenitor cell with repopulative abilities restricted to myelopoiesis and erythropoiesis. In other cases, the pluripotent progenitor cells in MDS may show the capacities to differentiate into B lineage lymphoid cells, as well as suggesting that in those instances MDS represents a condition of more primitive transformed hematopoietic ancestor cells.

The development of transplant coronary artery disease after cardiac transplantation is correlated with a predominance of CD8+ T lymphocytes in endomyocardial biopsy derived T cell cultures.

Long-term survival of heart transplant recipients is limited by the development of transplant coronary artery disease (TCAD). We analysed whether the development of TCAD is correlated with the incidence of acute rejection episodes, with the formation of anti-HLA antibodies or with the composition and function of T lymphocyte cultures derived from endomyocardial biopsies. TCAD was assessed by visual analysis of annually performed coronary angiograms and defined as the presence of all vascular changes, including minor wall irregularities. One year after transplantation, 31 of the 77 patients studied had TCAD (40%). The median age and mean number of HLA mismatches in patients with or without TCAD were highly comparable. The patient groups did not differ in incidence of acute rejection episodes, nor in percentage of endomyocardial biopsies yielding T cell cultures. At 1 year after transplantation, lymphocyte cultures from 18/31 TCAD+ patients (58%) and 27/46 TCAD- patients (57%) were analysed. The TCAD+ patients had, compared with the TCAD- patients, a higher median percentage of CD8+ T cells (71% versus 25%, P = 0.06) and a lower median percentage of CD4+ T cells (4% versus 40%, P = 0.04). Similar differences were found in a longitudinal analysis of the culture results of endomyocardial biopsies (EMBs) obtained during the first year. The cytotoxic reactivity of the cultures against donor HLA class I or class II antigens was comparable in the two groups, although a difference in recognition of heart specific antigens remains possible. The fact that EMB-derived cultures from TCAD+ and TCAD- patients differed in T cell phenotype populations gives some support to the hypothesis that cellular immunological processes are involved in the development of TCAD. However, while the median values differed, the overlap of the percentages of CD8+ cells in cultures from TCAD- and TCAD+ patients shows that other factors besides CD8+ T cells also play a role.

Long-term survival of heart grafts in the presence of donor-specific cytotoxic T-cell precursors (CTLp) in the peripheral blood.

To monitor their immunological status we determined donor and third-party-specific cytotoxic T-cell precursor frequencies (CTLpf) in the peripheral blood of 15 heart transplant recipients. PBL samples were obtained at different time points before and after transplantation. Donor-specific CTLpf and third-party-specific CTLpf were within the same range for all samples (1-1489/10(6) cells). The donor-specific CTLpf were not different between patients who had never had an acute rejection (AR) and patients who had an acute rejection as diagnosed by endomyocardial biopsy (EMB). No difference was observed between donor-specific CTLpf of samples taken on the day of transplantation and those obtained between 3 months and 3 years after transplantation. There was also no relationship between the donor-specific CTLpf in the PBL and the culturing success of lymphocytes from EMB taken at the same time. CTLpf were in the same range both when cultures could be propagated from the graft and when no cells grew out. We conclude that long-term graft survival is possible in the presence of CTLpf in peripheral blood.

Altered specificity of alloreactive cardiac graft-infiltrating cells by prophylactic treatment with OKT3 or horse antilymphocyte globulin.

Graft-infiltrating lymphocytes from patients who were prophylactically treated with OKT3 or horse antilymphocyte globulin (H-ALG) were found to have different specificity patterns from those in the control group that received cyclosporine from the day of transplantation. This prophylactic treatment led to a significant decrease of the HLA-DR-directed cytotoxicity, while the cytolytic response against HLA-class I mismatches was hardly affected. In H-ALG patients without rejection, the percentages of class I-reactive cultures were found to be lower than in the other treatment groups, which was mainly due to a lower percentage of HLA-B--reactive cultures. In CsA and OKT3 patients, cytotoxic T cells were rather directed to HLA-B mismatches than to HLA-A antigens, while in H-ALG patients no difference in HLA-A and B-directed cytotoxicity was found. Our data suggest that OKT3 and H-ALG influence the specificity of the T cell allorepertoire, resulting in a decreased frequency of class II-specific cytotoxic T cells after transplantation. H-ALG also has a downregulating influence on the CTL response against HLA class I (HLA-B) antigens. In some patients a fast regeneration of these cells occurs, which results in a higher rejection incidence during the first posttransplant year.

Phenotype of endomyocardial biopsy-derived T-lymphocyte cultures and chronic rejection after heart transplantation.

Chronic rejection (CR) is a major problem in long-term survival in heart transplantation. We analysed whether the occurrence of CR correlates with the incidence of acute rejections (AR) or with characteristics of endomyocardial biopsy-derived cell cultures. CR was diagnosed by annual angiography and defined as all coronary vascular changes. One year after transplantation 24 of the 63 patients had CR (38%). The incidence of AR in CR+ and CR- patients was comparable. The patients in both groups had similar individual median percentages of EMB-yielding cell cultures. During the first year the CR- patients had more cultures in which at least 60% of the cells were CD4+ T cells (50% vs 37%, P = 0.05), due to a stronger CD4 predominance in the first 6 months. In the second year the CD4 predominance in the patients diagnosed as CR+ after 1 year tended to be higher (P = 0.08). The patients had comparable percentages of cultures predominated by CD8+ T cells, gammadelta T cells or NK cells, irrespective of the time interval. These results might indicate that CD4+ T lymphocytes play a dual role in the aetiology of CR.

Donor directed cytotoxicity of cardiac graft infiltrating cells during cytomegalovirus infection.

We investigated whether cytomegalovirus (CMV) infection had an effect on donor directed cytotoxicity of cardiac graft infiltrating cells. The group we studied comprised 89 heart transplant recipients. Thirty eight showed signs of CMV infection, and in 27 of them cytolytic activity of biopsy-derived cultures could be tested during the infection. Fifty-one patients had never had CMV infection, and they were used as the control group. Eight patients had a primary, and 19 a secondary infection. We found that during CMV infection, both primary and secondary, a significantly higher proportion of the biopsy-derived cultures showed cytotoxicity against donor antigens (P < 0.01 when compared to the control group). In secondary infections, this was only due to an increase in donor class I directed cytotoxicity, while in primary infections a significant increase of class II directed cytotoxicity was also found (P < 0.005 when compared to secondary infection).

Changes in the activities of key enzymes of glycolysis during the cell cycle in yeast: a rectification.

Activities of glycolytic enzymes were determined in elutriation fractionated cultures of Saccharomyces cerevisiae grown on different carbon sources. Almost pure fractions of single cells at the G1 state of cell division were obtained for some of the growth conditions tested, whereas other stages were enriched in particular fractions. Specific activities of glucose-6-phosphate dehydrogenase and alcohol dehydrogenase were found to be constant during the cell cycle, as reported by van Doorn et al. (1988a), Journal of Bacteriology 170, 4808-4815, and (1988b), Journal of General Microbiology 134, 785-790. In contrast to the earlier reports, the activities of hexokinase, phosphofructokinase, pyruvate kinase and trehalase were also constant in different states of the cell cycle. For hexokinase and phosphofructokinase it was shown that the apparent specific activity in a cell-free extract strongly diminished when extracts contained less that 0.5-1 mg protein ml-1. In the experiments of van Doorn et al. (1988a) the protein content of the outer fractions was up to 20 times lower than that of the central fractions, suggesting an alternative explanation for the observed changes in enzyme activities during the cell cycle. Therefore, we want to rectify the observations presented by van Doorn et al. (1988a), and conclude that the activities of the glycolytic enzymes do not vary greatly during the cell cycle of S. cervisiae.

Haemophilus influenzae infections in patients with chronic obstructive pulmonary disease despite specific antibodies in serum and sputum.

The titer and specificity of antibodies to the infecting Haemophilus influenzae was determined in sera and sputa from 27 patients with chronic obstructive pulmonary disease (COPD) to analyze the specific immune response. COPD patients had significantly higher serum IgG and IgA antibody titers than 13 healthy controls (mean IgG titers 12,302 and 5,623, respectively; mean IgA titers, 2,398 and 912; p less than 0.001). The mean IgM titers were comparable: 501 and 447, respectively. Specific IgA antibodies were also detectable in the sputum of the COPD patients (mean IgA antibody titer, 776). The local antibody production was determined by calculating the relative coefficient of excretion (RCE) to albumin. The mean RCE of 89.1 for IgA indicated statistically significant local production (p less than 0.02), in contrast to a nonsignificant increase for IgG (mean RCE of 3.6). Specific IgM was below the detection level. Immunoblotting experiments showed that the antibodies in sera from COPD patients and controls were directed against most of the outer membrane proteins of H. influenzae, with individual differences between IgG, IgA, and IgM. The IgA and IgG antibodies in serum had a similar specificity as those in sputum. The appearance or persistence of H. influenzae coincided with minor changes in antibody titer and specificity. From these results we conclude that COPD patients are infected with H. influenzae despite the presence of at least as many antibodies in sputum and serum as in controls and that these antibodies are directed against a variety of antigenic determinants of the infecting strain.

Endogenous and exogenous reinfections by Haemophilus influenzae in patients with chronic obstructive pulmonary disease: the effect of antibiotic treatment on persistence.

To analyze whether exacerbations in chronic obstructive pulmonary disease (COPD) coincide with reinfection by Haemophilus influenzae, 16 COPD patients were studied longitudinally for 3 years. Exacerbations coincided with reinfection by H. influenzae, either endogenous, by a strain with a DNA fingerprint indistinguishable from the strain previously present but with another major outer membrane protein (MOMP) pattern (2 patients), or exogenous, by a strain with a different DNA fingerprint and MOMP pattern (3 patients). The other patients, remaining in an infectious state without clear exacerbations for longer periods, were persistently infected by a particular H. influenzae strain (median persistence time, 5.5 months; range, 2-23 months). Of 8 antibiotic-treated patients, 7 remained infected by H. influenzae with the same DNA fingerprint, although all strains were sensitive to the antibiotics prescribed. Results of the study suggested that exacerbations in COPD patients coincide with endogenous or exogenous reinfection by H. influenzae, persistently infected patients keep the same H. influenzae strain for longer periods, and antibiotic treatment was not effective in eradicating H. influenzae.