Genotoxicity of cytokines at chemotherapy-induced 'storm' concentrations in a model of the human bone marrow.

Donor cell leukaemia (DCL) is a complication of haematopoietic stem cell transplantation where donated cells become malignant within the patient's bone marrow. As DCL predominates as acute myeloid leukaemia, we hypothesized that the cytokine storm following chemotherapy played a role in promoting and supporting leukaemogenesis. Cytokines have also been implicated in genotoxicity; thus, we explored a cell line model of the human bone marrow (BM) to secrete myeloid cytokines following drug treatment and their potential to induce micronuclei. HS-5 human stromal cells were exposed to mitoxantrone (MTX) and chlorambucil (CHL) and, for the first time, were profiled for 80 cytokines using an array. Fifty-four cytokines were detected in untreated cells, of which 24 were upregulated and 10 were downregulated by both drugs. FGF-7 was the lowest cytokine to be detected in both untreated and treated cells. Eleven cytokines not detected at baseline were detected following drug exposure. TNFα, IL6, GM-CSF, G-CSF, and TGFß1 were selected for micronuclei induction. TK6 cells were exposed to these cytokines in isolation and in paired combinations. Only TNFα and TGFß1 induced micronuclei at healthy concentrations, but all five cytokines induced micronuclei at storm levels, which was further increased when combined in pairs. Of particular concern was that some combinations induced micronuclei at levels above the mitomycin C positive control; however, most combinations were less than the sum of micronuclei induced following exposure to each cytokine in isolation. These data infer a possible role for cytokines through chemotherapy-induced cytokine storm, in the instigation and support of leukaemogenesis in the BM, and implicate the need to evaluate individuals for variability in cytokine secretion as a potential risk factor for complications such as DCL.

IL-6 knockdown in a model of the human bone marrow, abrogates DNA damage induction in bystander cells post-chemotherapy induced cytokine release syndrome.

Following infection or exposure to therapeutic agents, an aggressive immune response may result, termed cytokine storm (CS) or cytokine release syndrome. Here the innate immune system becomes uncontrolled, leading to serious consequences including possible death. Patients surviving CS are at greater risk for de novo tumorigenesis, but it is unclear if any specific cytokines are directly responsible for this outcome. De novo tumorigenesis has been observed in donated cells exposed to CS following haematopoietic stem cell transplant (HSCT). Modelling HSCT, we firstly demonstrated the release of CS levels from the HS-5 human bone marrow stromal cell line, post-exposure to chemotherapy. We then exposed the TK6 lymphoblast cell line to healthy and storm doses of IL-6 and measured increased genotoxicity via the micronucleus assay. During HSCT, haematopoietic cells are exposed to a complex mix of cytokines, so to determine if IL-6 was integral in a chemotherapy-induced bystander effect, we attempted to inhibit IL-6 from HS-5 cells using resatorvid or siRNA, treated with chlorambucil or mitoxantrone, and then co-cultured with bystander TK6 cells. Whilst resatorvid did not reduce IL-6 and did not reduce micronuclei in the bystander TK6 cells, siRNA inhibition reduced IL-6 to healthy in vivo levels, and micronuclei aligned with untreated controls. Our data suggests that exposure to high IL-6 (in the absence of inflammatory cells) has potential to induce genetic damage and may contribute to de novo tumorigenesis post-CS. We suggest that for individuals with a pro-inflammatory profile, anti-IL-6 therapy may be an appropriate intervention to prevent complications post-CS.

Comparative study of male and female human hair: A microscopic analysis.

The outer cuticle, middle cortex, and inner medulla make up hair, which is an epidermal outgrowth. Hair is resilient under harsh natural conditions, thus it is frequently collected at crime scenes, making human hair analysis important in the forensic sciences field. It aids in the formation of a triangle connecting a crime scene, a victim, and a culprit. The aim of this study is to observe the microscopic structure of male and female human hair. Samples of hair specimens from males and females were collected. The materials used were ethanol to degrease and a stereomicroscope to observe the structural differences between the male and female hair samples. The comparison between male and female hair is done on the grounds of color, shaft profiles, the proximal and distal ends of the hair, cuticle, and surface texture, and the other found characters. This study of comparison between male and female hair specimens revealed that the hair color at the distal end is found to be brown for females while it is completely black in that of males, and the surface texture of males is found to have some irregularities while there are no irregularities in female. This study can be concluded that the structural comparison between male and female hair specimens can be used as evidence for forensic analysis at crime scenes.

Insulin Usage and Practices in Children and Adolescents with Type 1 Diabetes.

CONTEXT: Data on insulin usage and practices in children and adolescents with type 1 diabetes (T1D) is sparse in India. AIMS: To analyze the various insulin types and regimens used by children and adolescents with T1D, the techniques and the devices used for insulin administration, and the storage and disposal methods of delivery devices. SETTINGS AND DESIGN: Observational cross-sectional study. METHODS AND MATERIALS: Study subjects were children and adolescents with T1D ≥6 months and informed consent was obtained. A detailed demographic history was collected, and a predesigned, pretested questionnaire was used. RESULTS: The number of subjects were 90 (M: F; 32:58), age ranging from 3 to 18 years and duration of T1D was 6 months to 16 years. Mean age was 13 ± 4.6 yrs, HbA1c was 9.11 ± 2.2% and duration was 5 years. Conventional insulins were more commonly used than analogs. Basal-bolus (BB) regimen was used in 49% of the subjects. Mean HbA1c for analogs was 7.6% and conventional was 9.3% (p = 0.001). HbA1c <8% was significantly more in those aged 3-8 yrs, mean duration ≤4.1 yrs, those using pens and BB regimen. Fifty-six percent were using own refrigerators for storage and the most common barriers for insulin usage were fear of hypoglycemia (37%), inaccessibility (20%), and apprehension of shots (18%). Site rotation patterns were followed by 84% and 94% of the subjects reported disposing syringes and sharps as general waste. CONCLUSIONS: Conventional insulins and vial-syringes remain the most commonly used insulin delivery systems. Glycemic control was better in younger age, lesser duration, BB regimen, analog usage, and pen devices.

In vitro release of digestive enzymes by FMRF amide related neuropeptides and analogues in the lepidopteran insect Opisina arenosella (Walk.).

The insect neuropeptides FMRF amide, leucomyosupressin (LMS) and neuropeptide analogues leucosulfakinins (FLSK and LSK II Ser (SO(3)H)), perisulfakinin (PSK), proleucosulfakinin (PLSK), 14A[phi1]WP-I, 542phi1, and 378A[5b]WP-I were assayed for their effects on the release of amylase and protease from the midgut tissue of larvae of Opisina arenosella. In the bioassay, empty midgut tubes ligated at both ends using hair were incubated with insect saline containing neuropeptides/analogues in a bioassay apparatus at 37 degrees C for 30 min. After incubation the contents of the midgut preparations were analyzed for amylase and protease activity. In control experiments, the midgut preparations were incubated in insect saline without neuropeptides. The results of the study reveal that for stimulating amylase release from midgut tissue, the peptides require an FXRF amide (X may be methionine or leucine) sequence at the C-terminal. The presence of HMRF amide at C-terminal of peptides may inhibit the release of amylase. Meanwhile, peptides with both FMRF and HMRF amide sequence at the C-terminal are found to be effective in stimulating protease release. The tetrapeptide segment at the C-terminal probably represent the active core of the neuropeptide.

A brain peptide stimulates release of amylase from the midgut tissue of larvae of Opisina arenosella Walk. (Lepidoptera: Cryptophasidae).

Brain extracts from 3 to 4 day old final (eighth) instar larvae of Opisina arenosella (Lepidoptera) stimulate amylase release from midgut preparations maintained in vitro. This effect of the brain extract was both time and dose dependent. The brain factor stimulating enzyme release may be a peptide as it is heat stable and susceptible to treatment with proteolytic enzymes. For purification of the brain factor, a head extract prepared in 2% NaCl was first precipitated in 80% aqueous acetone and then fractionated by DEAE cellulose ion exchange chromatography. The fraction OCF(2), from ion exchange chromatography was further purified on a Sephadex G25 column. The fraction designated as OCF(2.3) obtained by gel filtration showed maximum activity and it was selected for HPLC analysis. HPLC elution profiles of OCF(2.3) showed two major peaks separated by a time interval of 0.107 min. The two overlapping peaks of OCF(2.3) may represent either different forms of a peptide or different peptides of a family. The molecular weight OCF(2.3) was estimated to be 1070 Da.

Inhibition of digestive enzyme release by neuropeptides in larvae of Opisina arenosella (Lepidoptera: Cryptophasidae).

Leucokinins are a group of structurally related neuropeptides stimulating gut motility and fluid secretion by Malpighian tubule in insects. For studying effect of neuropeptides on digestive enzyme release, empty midgut tubes of larvae of Opisina arenosella ligated at both ends with hair were incubated with Leucokinins (LK I-VIII), LK analogues and Leucopyrokinin (LPK) in a bioassay apparatus at 37 degrees C for 30 min. The lumen contents were subsequently analyzed for digestive enzyme levels. The neuropeptides LK III, FFSWG amide, 122 A[1] WP-2, LPK and 434 [phi2] WP-1 inhibited the release of digestive enzymes, protease and amylase while LK VIII, unique in having tyrosine residue, stimulated protease release. The minimum sequence of amino acids at the C-terminal required for activity of LK peptides was found to be FXSWGamide (X=Asn, His, Ser, or Trp). The N-terminal pyroglutamate residue and proline at the C-terminal may contribute to the inhibitory effect of LPK on digestive enzyme release. The present study reveals for the first time an inhibitory effect for leucokinins and pyrokinin on the release of digestive enzymes from the insect midgut.