Influenza A and B Clinical Trial
Official title:
Sub Populations of Immune Cells in Influenza A and B Patients
This study designed to examine changes of immune system cells sub-populations during
influenza disease. Several parameters will be examined, such as: amount of sub populations,
clinical parameters (body temperature and number of hospitalization days).
The participants are children that are hospitalized in the Laniado hospital pediatric
department.
Background:
In late March and early April 2009 an outbreak of a novel influenza virus designated A/H1N1
influenza (popularly known as swine flu) was reported in Mexico, with subsequent cases
observed in many countries, leading to the declaration by the World Health Organization of a
worldwide pandemic.
The first case of influenza A/H1N1 in Israel was diagnosed at the end of April 2009 at
Laniado Hospital in a young man returning from Mexico.
Influenza A/H1N1 (Swine flu) carries a relatively high morbidity, particularly in young
patients. Early identification would enable prompt initiation of therapy, thereby improving
outcomes.
In 2009, during the H1N1 pandemic in Israel, a research was conducted in our department of
pediatrics. The researchers Sharon et al. (1) have compared a group of 53 children infected
with Influenza A H1N1, with a group of 53 children that presented with flu symptoms but were
found negative for the virus. This comparison demonstrated a significant reduction in
lymphocyte count during the acute phase of the disease (days 1-3) with resolution on day 7
(6-9) as well as reduction in the neutrophil count on day 5 from the onset of high fever with
subsequent resolution 2-4 days later (days 7-10). This phenomenon was not observed in the
patients that were not infected with the H1N1 virus. Similar results were reported by Cao and
co-authors (2) that observed it in both pediatric and adult patients. Lewis at al suggested
that the lymphopenia is mainly due to reduction in T cells and to a lesser extent in B cells
(3). The lymphopenia typical of influenza A H3N3 during acute illness was shown to be due to
a reduction in both T and B cells without alteration in the CD4:CD8 ratio. This is in
contrast to the findings of Cao et al. (2) who noted an abnormal CD4:CD8 ratio in half of
those who were positive for A H1N1. One of the possible explanations as proposed by Nichols
and Collaborators (4) is that early lymphopenia and the later neutropenia in the influenza
infected patients may represent migration of these cells from the circulation to the infected
respiratory tract as a consequence of infection. Goals of our research To quantify the B, T
and NK populations of lymphocytes during different stages of the disease caused by Infuenza A
H1N1.
Study methods The subjects of the study were children that were hospitalized at our
department with signs and symptoms of flu. The parents or legal guardians of the participants
have signed a written consent. At the first stage an upper respiratory swab was taken to
determine the presence of Infuenza A H1N1 (the virus identification was done by RT - PCR).
Then the different subtypes of lymphocytes were quantified at different stages of the
disease. The identification and quantification of the different cell types were performed by
FACS method, the cell receptors that were marked were CD31, CD19, CD4, CD3, CD45, CD8, CD56.
Laboratory technique:
Cellular markers were assessed by flow cytometry. Cell staining was performed on single cell
suspensions using pre-cooled PBS in 5 ml FACS tubes. Washing steps were performed by
centrifugation for 5min, 300 g, 5°C, brake 5 after which supernatants were completely
pipetted off and cell pellets (typically containing 0.2-1.0×106 cells) were re-suspended in
100 μl PBS (without Ca++and Mg++) supplemented with 0.5% bovine serum albumin, stained with
specific fluorochrome-conjugated anti-human antibodies or isotype-matched non-specific
controls and incubated in the refrigerator 2-8°C for 30 min. After incubation, cells were
washed by centrifugation and re-suspended in 0.25-0.5 ml cold PBS.
Cells tested were stained with mouse monoclonal antibodies for CD45-FITC, CD45-PE a pan
leukocyte marker, T Cell marker CD3-PE CD8-FITC, T helper cells CD4-FITC, CD8-PE, Natural
killer (NK) cell marker CD56-PE, monocyte marker CD31-FITC and B cell marker CD19-PE.
Emission compensation was done by positive control tubes containing cells that were stained
with CD45-FITC or CD45-PE; Dead cells were excluded by staining with 7-aminoactinomycin D,
only un-stained viable cells were analyzed. At least 10,000 cellular events per sample were
analyzed using the CellQuest Pro or FlowJo software. Percentages of isotype control antibody
staining, not exceeding 1% stained cells, were used to set the 'positive'staining gates, and
were deduced from the specific antibody results. The results are expressed as the percentage
of stained cells.
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