Challenges faced by ethics committee members in India during COVID-19 pandemic: A mixed-methods exploration.

Background & objectives: The COVID-19 pandemic had a distinct impact on scientific research and Ethics Committees (ECs). We conducted a mixed-methods investigation to understand the issues faced and solutions identified by ECs during this pandemic in India. Methods: A quantitative online survey form (30 members) and qualitative in-depth interviews (10 members) from various ECs were conducted. Thematic content analysis for qualitative and proportion analysis for quantitative data was carried out. Results: During the online survey, an average difficulty score, which was measured using the Visual Analogue Scale, was 5.3 (SD 2.1). Pressure for expedited approvals was felt by EC members with a drastic increase in the number of submission of research projects. The scarcity of information on investigational products (IPs) and requisite consent process posed major hurdles. Ongoing non-COVID studies and post-graduate dissertations were badly hit due to the shift in attention towards COVID-related research. Non-familiarity with virtual technology and lack of face-to-face interactions were highlighted as demerits. However, a few of the EC members welcomed newer methods, being time-saving, convenient and reducing travel hassles. Site monitoring and severe adverse event-related analyses were also negatively impacted upon. Solutions included the alternate methods of consenting (virtual, abbreviated), a detailed explanation of the protocol and IPs and benefits versus risk assessment. Interpretation & conclusions: Despite various challenges posed by the COVID-19 pandemic, the ECs in India steered well through the hurdles. Moreover, adapting a hybrid mode, technical training and updating guidelines were perceived as urgent by EC members.

Generation of Genetically Engineered Precursor T-Cells From Human Umbilical Cord Blood Using an Optimized Alpharetroviral Vector Platform.

Retroviral engineering of hematopoietic stem cell-derived precursor T-cells (preTs) opens the possibility of targeted T-cell transfer across human leukocyte antigen (HLA)-barriers. Alpharetroviral vectors exhibit a more neutral integration pattern thereby reducing the risk of insertional mutagenesis. Cord blood-derived CD34+ cells were transduced and differentiated into preTs in vitro. Two promoters, elongation-factor-1-short-form, and a myeloproliferative sarcoma virus variant in combination with two commonly used envelopes were comparatively assessed choosing enhanced green fluorescent protein or a third-generation chimeric antigen receptor (CAR) against CD123 as gene of interest. Furthermore, the inducible suicide gene iCaspase 9 has been validated. Combining the sarcoma virus-derived promoter with a modified feline endogenous retrovirus envelope glycoprotein yielded in superior transgene expression and transduction rates. Fresh and previously frozen CD34+ cells showed similar transduction and expansion rates. Transgene-positive cells did neither show proliferative impairment nor alteration in their lymphoid differentiation profile. The sarcoma virus-derived promoter only could express sufficient levels of iCaspase 9 to mediate dimerizer-induced apoptosis. Finally, the CD123 CAR was efficiently expressed in CD34+ cells and proved to be functional when expressed on differentiated T-cells. Therefore, the transduction of CD34+ cells with alpharetroviral vectors represents a feasible and potentially safer approach for stem cell-based immunotherapies for cancer.

Endoxifen, a new cornerstone of breast cancer therapy: demonstration of safety, tolerability, and systemic bioavailability in healthy human subjects.

Endoxifen is an active metabolite of tamoxifen, a drug used in the treatment of breast cancer. In order to be clinically effective, tamoxifen must be converted to endoxifen by cytochrome P450 2D6 (CYP2D6). A study involving single escalating oral doses was conducted in humans to evaluate the safety, tolerability, and pharmacokinetics (PK) of endoxifen. This is the first study demonstrating that single oral doses of endoxifen are safe and well tolerated and have sufficient bioavailability to reach systemically effective levels in human subjects. Furthermore, it was found that endoxifen is rapidly absorbed and systemically available and that it displays dose proportionality in peak drug concentrations in plasma (C(max)) and area under the concentration-time curve extrapolated from 0 to ∞ (AUC(0-∞)) over the dose range 0.5-4.0 mg.

Expression of the C-C chemokine receptor CCR3 in human airway epithelial cells.

Chemokine-induced eosinophil chemotaxis is mediated primarily through the C-C chemokine receptor, CCR3. We have now detected CCR3 immunoreactivity on epithelial cells in biopsies of patients with asthma and other respiratory diseases. CCR3 mRNA was detected by Northern blot analysis after TNF-alpha stimulation of the human primary bronchial epithelial cells as well as the epithelial cell line, BEAS-2B; IFN-gamma potentiated the TNF-alpha-induced expression. Western blots and flow cytometry confirmed the expression of CCR3 protein. This receptor is functional based on studies demonstrating eotaxin-induced intracellular Ca(2+) flux and tyrosine phosphorylation of cellular proteins. The specificity of this functional response was confirmed by blocking these signaling events with anti-CCR3 mAb (7B11) or pertussis toxin. Furthermore, (125)I-eotaxin binding assay confirmed that CCR3 expressed on epithelial cells have the expected ligand specificity. These studies indicate that airway epithelial cells express CCR3 and suggest that CCR3 ligands may influence epithelial cell functions.

Effect of gender on the development of hypocapnic apnea/hypopnea during NREM sleep.

We hypothesized that a decreased susceptibility to the development of hypocapnic central apnea during non-rapid eye movement (NREM) sleep in women compared with men could be an explanation for the gender difference in the sleep apnea/hypopnea syndrome. We studied eight men (age 25-35 yr) and eight women in the midluteal phase of the menstrual cycle (age 21-43 yr); we repeated studies in six women during the midfollicular phase. Hypocapnia was induced via nasal mechanical ventilation for 3 min, with respiratory frequency matched to eupneic frequency. Tidal volume (VT) was increased between 110 and 200% of eupneic control. Cessation of mechanical ventilation resulted in hypocapnic central apnea or hypopnea, depending on the magnitude of hypocapnia. Nadir minute ventilation in the recovery period was plotted against the change in end-tidal PCO(2) (PET(CO(2))) per trial; minute ventilation was given a value of 0 during central apnea. The apneic threshold was defined as the x-intercept of the linear regression line. In women, induction of a central apnea required an increase in VT to 155 +/- 29% (mean +/- SD) and a reduction of PET(CO(2)) by -4.72 +/- 0.57 Torr. In men, induction of a central apnea required an increase in VT to 142 +/- 13% and a reduction of PET(CO(2)) by -3.54 +/- 0.31 Torr (P = 0.002). There was no difference in the apneic threshold between the follicular and the luteal phase in women. Premenopausal women are less susceptible to hypocapnic disfacilitation during NREM sleep than men. This effect was not explained by progesterone. Preservation of ventilatory motor output during hypocapnia may explain the gender difference in sleep apnea.

Cutaneous injection of human subjects with macrophage inflammatory protein-1 alpha induces significant recruitment of neutrophils and monocytes.

Macrophage inflammatory protein (MIP-1 alpha), a member of the CC chemokine subfamily, has been shown to attract T cells and monocytes in vitro and to be expressed at sites of inflammation. Although the in vitro activities of MIP-1 alpha have been well documented, the in vivo biological activities of MIP-1 alpha in humans have not been studied. To address this, we challenged human subjects by intradermal injection with up to 1000 pmol of MIP-1 alpha and performed biopsies 2, 10, and 24 h later. Although no acute cutaneous or systemic reactions were noted, endothelial cell activation, as indicated by the expression of E-selectin, was observed. In agreement with its in vitro activity, monocyte, lymphocyte, and, to a lesser degree, eosinophil infiltration was observed, peaking at 10-24 h. Surprisingly, in contrast to its reported lack of in vitro neutrophil-stimulating activity, a rapid infiltration of neutrophils was observed in vivo. This neutrophil infiltration occurred as early as 2 h, preceding the appearance of other cells, and peaked at 10 h. Interestingly, we found that neutrophils in whole blood, but not after isolation, expressed CCR1 on their cell surface. This CCR1 was thought to be functional as assessed by neutrophil CD11b up-regulation following whole-blood MIP-1 alpha stimulation. These studies substantiate the biological effects of MIP-1 alpha on monocytes and lymphocytes and uncover the previously unrecognized activity of MIP-1 alpha to induce neutrophil infiltration and endothelial cell activation, underscoring the need to evaluate chemokines in vivo in humans.

Migration of eosinophils across endothelial cell monolayers: interactions among IL-5, endothelial-activating cytokines, and C-C chemokines.

Eosinophils are the predominant cell type recruited in inflammatory reactions in response to allergen challenge. The mechanisms of selective eosinophil recruitment in allergic reactions are not fully elucidated. In this study, the ability of several C-C chemokines to induce transendothelial migration (TEM) of eosinophils in vitro was assessed. Eotaxin, eotaxin-2, monocyte chemotactic protein (MCP)-4, and RANTES induced eosinophil TEM across unstimulated human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner with the following rank order of potency: eotaxin approximately eotaxin-2 > MCP-4 approximately RANTES. The maximal response induced by eotaxin or eotaxin-2 exceeded that of RANTES or MCP-4. Preincubation of eosinophils with anti-CCR3 Ab (7B11) completely blocked eosinophil TEM induced by eotaxin, MCP-4, and RANTES. Activation of endothelial cells with IL-1beta or TNF-alpha induced concentration-dependent migration of eosinophils, which was enhanced synergistically in the presence of eotaxin and RANTES. Anti-CCR3 also inhibited eotaxin-induced eosinophil TEM across TNF-alpha-stimulated HUVEC. The ability of eosinophil-active cytokines to potentiate eosinophil TEM was assessed by investigating eotaxin or RANTES-induced eosinophil TEM across resting and IL-1beta-stimulated HUVEC in the presence or absence of IL-5. The results showed synergy between IL-5 and the chemokines but not between IL-5 and the endothelial activator IL-1beta. Our data suggest that eotaxin, eotaxin-2, MCP-4, and RANTES induce eosinophil TEM via CCR3 with varied potency and efficacy. Activation of HUVEC by IL-1beta or TNF-alpha or priming of eosinophils by IL-5 both promote CCR3-dependent migration of eosinophils from the vasculature in conjunction with CCR3-active chemokines.

Age-related changes in blood lymphocyte subsets of Saudi Arabian healthy children.

The age-related changes in absolute and percentage values of lymphocyte subsets in the peripheral blood of healthy children of different ages (1 month to 13 years) were studied by flow cytometry. The absolute and percentage values for most lymphocyte subpopulations differed substantially with age. Comparisons among age groups from infants through adults revealed progressive declines in the absolute numbers of leukocytes, total lymphocytes, and T, B, and natural killer (NK) cells. The percentages of T cells increased with age. Within the T-lymphocyte population, the CD8(+) subset increased but the CD4(+) subset decreased, resulting in a declining CD4(+)/CD8(+) ratio. The percentage of B cells declined, but that of NK cells remained unchanged. The percentage of HLA-DR+ T cells increased over time, but their number changed inconsistently. Our findings confirm and extend earlier reports on age-related changes in lymphocyte subpopulations. These data should be useful in the interpretation of disease-related changes, as well as therapy-dependent alterations, in lymphocyte subsets in children of different age groups.

Lymphocyte subset reference ranges in healthy Saudi Arabian children.

In an attempt to establish the reference ranges for lymphocyte subsets in children, the distribution of lymphocyte population-bearing surface markers such as CD3 (T cells), CD19 (B cells), CD4 (T helper/inducer cells), CD8 (T suppressor/cytotoxic cells), and CD16 and/or CD56 on CD3- cells (NK cells) has been studied among healthy Saudi Arabian infants and children. Normal adult blood donors were used for comparison. Anticoagulated peripheral blood was stained with monoclonal antibodies and the lymphocytes were analyzed by flow cytometry for the expression of the above markers. Absolute and percentage values for most lymphocyte populations differed substantially not only between children and adults but also among children from different age groups. Absolute numbers of all the lymphocyte subsets decreased with age from 1 month to 13 years; the median value declined from 4.1 to 1.9 (T cells), 1.6 to 0.6 (B cells), 0.5 to 0.3 (NK cells), 2.7 to 1.0 (CD4+ T cells) and 1.5 to 0.8 x 10(3) cells/mm3 (CD8+ T cells). HLA-DR+ T cell counts changed significantly from 0.3 to 0.2 x 10 (3) cells/mm3 during the same age period. In contrast, the lymphocyte percentage increased in all the subsets except B cells and CD4+ T cells with time. The percentage values increased from 66 to 74 (T cells), 8 to 11 (NK cells), 23 to 39 (CD8+ T cells) and 4 to 9 (HLA-DR+ T cells). The values changed from 24 to 12 and 46 to 39 for B cells and CD4+ T cells, respectively, with age from 1 month to 13 years. The variations in CD4+ and CD8+ T cells resulted in a decrease in CD4+/CD8+ ratio from 2.0 to 1.1 with age. These data should be useful as reference values for lymphocyte subsets in various diseases of infants and children.

Quantitative differences in CD8+ lymphocytes, CD4/CD8 ratio, NK cells, and HLA-DR(+)-activated T cells of racially different male populations.

In an attempt to establish the reference ranges for lymphocyte subsets the distribution of lymphocyte-population-bearing surface markers such as CD3 (T cells), CD19 (B cells), CD4 (T helper/inducer cells), CD8 (T suppressor/cytotoxic cells), HLA-DR on CD3+ cells (activated T cells), and CD16 and/or CD56 on CD3- cells (NK cells) has been studied among normal male Saudi blood donors. Anticoagulated peripheral blood was stained with monoclonal antibodies and the lymphocytes were analyzed by flow cytometry for the expression of the above markers. Reference ranges were as follows: CD3+ (61 to 87%), CD19+ (5 to 27%), CD4+ (31 to 55%), CD8+ (24 to 56%), CD4+/CD8+ ratio (0.5 to 1.8), CD3+ HLA-DR+ (4 to 29%), and CD3- CD16+/CD56+ (4 to 29%). Furthermore, the absolute cell numbers for each lymphocyte subset are reported here for the first time for the Saudi male population. The most significant finding of the present study is the presence of a higher percentage and number of CD8+ T cells (P < 0.01) and a decreased CD4/CD8 ratio (P < 0.02) compared with the Caucasian controls. In addition, the Saudi male population has a significantly lower percentage and number of activated T cells (P < 0.05 and < 0.01, respectively) and a lower number of NK cells (P < 0.001).

Expression of IL-2 receptor subunit p55 (CD25) on normal human lymphocytes.

Unstimulated peripheral blood lymphocytes of normal Saudi Arabian males were examined for the expression of IL-2 receptor subunit p55 (IL-2Rp55) by two-color flow cytometric analysis using monoclonal antibodies. Eighteen percent of the normal lymphocytes were positive for this marker. Approximately 84% of the IL-2Rp55+ cells were CD3+ T cells of which CD4+ and CD8+ T cell subsets constituted 75 and 13%, respectively. CD19+ B cells made up 12% of the IL-2Rp55+ lymphocytes. In the pan T cell population 20% cells were IL-2Rp55+, whereas among the B cells 16% cells were IL-2Rp55+. In the CD4+ and CD8+ T cell population IL-2Rp55+ cells were 29 and 7% respectively. In comparison, normal Caucasian male population had significantly higher proportions of IL-2Rp55+ lymphocyte subsets, the total IL-2Rp55+ lymphocytes being 33 +/- 10%. However, except for CD8+ T cells, there was no difference in the mean phenotypic values of IL-2Rp55+ cells between the two populations. The present findings clearly show a significant expression of IL-2Rp55 (CD25) on peripheral blood lymphocytes in normal, healthy Saudi Arabian blood donors and Caucasians. It is suggested that the baseline values of IL-2Rp55+ lymphocytes must be established for the local normal population before the expression of this marker is implicated either as an active immune response or as an immune disorder.

Expression and release of IL-2 receptor and production of IL-2 by activated T lymphocyte subsets.

T lymphocyte subsets differ in expression of cell surface antigen and functional properties. Both CD4+ and CD8+ subsets express interleukin-2 receptor (IL-2R) following their activation in vitro. In the present investigation T lymphocyte subsets were activated by different mitogens and IL-2R expression was enumerated on these stimulated subsets. Peripheral blood mononucleated cells (PBMC) were stimulated with phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) and then stained with anti-CD4 or anti-CD8 antibodies conjugated with fluorescein isothiocyanate and anti-IL-2R monoclonal antibody conjugated with phycoerythrin using a direct immunofluorescence technique. The percentage of IL-2R positive lymphocytes was enumerated by flow cytometry. The results showed that mitogen activated lymphocytes expressed variable degrees of IL-2R which were significantly higher than the control. 53% of CD4+ lymphocytes and 28% of CD8+ expressed IL-2R following PHA stimulation in vitro. Similarly, 47% of CD4+ lymphocytes and 23% of CD8+ lymphocytes expressed IL-2R following PWM stimulation. The present study also revealed that the release of soluble IL-2R (sIL-2R) and IL-2 production in supernatant from cultured PBMC varied with different mitogen stimulation. Using the same concentration of PHA and PWM as used to study IL-2R expression, higher activity of sIL-2R was detected in PHA stimulated lymphocytes as compared to PWM treated lymphocytes. However, IL-2 production was more in culture medium from PMW treated PBMC. Thus, there was a significant correlation between the cellular and soluble IL-2R but the production of IL-2 from activated PBMC cells had no good correlation with either the cellular IL-2R expression or the release of sIL-2R.(ABSTRACT TRUNCATED AT 250 WORDS)

A study of angiogenesis factors from five different sources using a radioimmunoassay.

The use of a radioimmunoassay (RIA) demonstrated that angiogenic factors from human and animal tumours, normal bovine retinas, myocardial infarcts, synovial fluid from patients with joint diseases and wound fluid shared common antigenic determinants. Values for angiogenesis factors expressed as microgram Walker tumour TAF/mg protein varied from 0.8 (wound fluid) to 207 (myocardial infarct). Activated macrophages produced an angiogenic factor which did not cross-react. Normal tissue extracts which were non-angiogenic by the chicken chorioallantoic membrane assay also failed to cross-react in the RIA. The antigenic similarity of angiogenic factors from such a wide variety of sources suggests that, in order to minimize therapeutic side effects, it would be best to use angiogenic agonists and antagonists locally.

Tumour angiogenesis factor in urological cancers.

The presence of a tumour angiogenesis factor (TAF) was investigated by bioassay and radioimmunoassay (RIA) in tissue and urine from patients with a variety of urological cancers. TAF was present in the majority of tumours studied by both methods and absent from controls. Our results suggest that RIA of urine for TAF may be applicable as a screening test for bladder cancer.

Angiogenesis factor from human myocardial infarcts.

5 of 8 infarcted human myocardial tissues were shown to contain an angiogenesis factor which resembles that obtained from solid tumours. The concentration of angiogenesis factor, measured by radioimmunoassay, was similar to that in tumours. It is suggested that the myocardial-infarct angiogenesis factor modulates collateral enlargement or ingrowth of new blood vessels in the infarcted tissues.

Quantitation of angiogenesis factor in bovine retina and tumour extracts by means of radioimmunoassay.

Using an antiserum raised against tumour angiogenesis factor (TAF) we have developed a radioimmunoassay for retina and tumour angiogenesis factor(s). This antiserum was previously shown to bind to both human and animal tumour extracts and to inhibit the angiogenesis induced by TAF in vivo. TAF from rat Walker tumour was used for iodination by the chloramine-T method. An excess of 125I-labelled TAF was incubated with TAF antibody in the absence (maximum binding) and presence (inhibition of maximum binding) of unlabelled tissue extract. A double antibody technique was used to separate free and bound TAF. Unlabelled human Wilms tumour TAF was used as a standard. The extent of inhibition of 125I-TAF-anti-TAF binding provided a measure of TAF in tissue extracts examined. Extracts of normal bovine retina, cornea, lung, aorta, lymph nodes, iris, vitreous humour, and human tumours and normal human pituitary and liver were assayed. Only bovine retina and human tumours were found to contain angiogenesis factor. These findings, together with our earlier results, suggest that angiogenesis factor from both bovine retina and human tumours induce angiogenesis in vivo and possess common antigenic determinants. The presence of angiogenesis factor in healthy retina and its relationship to neovascularisation in clinical conditions is discussed.