Bone marrow stem cell therapy partially ameliorates pathological consequences in livers of mice expressing mutant human α1-antitrypsin.

Alpha-1-antitrypsin (AAT) deficiency (AATD) is a genetic disease, caused by mutation of the AAT gene. Accumulation of mutated AAT protein aggregates in hepatocytes leads to endoplasmic reticulum stress, resulting in impairment of liver functions and, in some cases, hepatocellular carcinoma, whereas decline of AAT levels in sera is responsible for pulmonary emphysema. In advanced liver disease, the only option for treatment is liver transplantation, whereas AAT replacement therapy is therapeutic for emphysema. Given that hepatocytes are the primary affected cells in AATD, we investigated whether transplantation of bone marrow (BM)-derived stem cells in transgenic mice expressing human AATZ (the Z variant of AAT) confers any competitive advantages compared to host cells that could lead to pathological improvement. Mouse BM progenitors and human mesenchymal stem cells (MSCs) appeared to contribute in replacement of 40% and 13% host hepatocytes, respectively. Transplantation of cells resulted in decline of globule-containing hepatocytes, improvement in proliferation of globule-devoid hepatocytes from the host-derived hepatocytes, and apparently, donor-derived cells. Further analyses revealed that transplantation partially improves liver pathology as reflected by inflammatory response, fibrosis, and apoptotic death of hepatocytes. Cell therapy was also found to improve liver glycogen storage and sera glucose level in mice expressing human AATZ mice. These overall improvements in liver pathology were not restricted to transplantation of mouse BM cells. Preliminary results also showed that following transplantation of human BM-derived MSCs, globule-containing hepatocytes declined and donor-derived cells expressed human AAT protein. CONCLUSION: These results suggest that BM stem cell transplantation may be a promising therapy for AATD-related liver disease. (Hepatology 2017;65:1319-1335).

Molecular and Cellular Functions Distinguish Superior Therapeutic Efficiency of Bone Marrow CD45 Cells Over Mesenchymal Stem Cells in Liver Cirrhosis.

Liver fibrosis is strongly associated with chronic inflammation. As an alternative to conventional treatments for fibrosis, mesenchymal stem cells (MSCs) therapy is found to be attractive due to its immunomodulatory functions. However, low survival rate and profibrogenic properties of MSCs remain the major concerns, leading to skepticism in many investigators. Here, we have asked the question whether bone marrow (BM)-derived CD45 cells is the better candidate than MSCs to treat fibrosis, if so, what are the molecular mechanisms that make such distinction. Using CCl4 -induced liver fibrosis mouse model of a Metavir fibrosis score 3, we showed that BM-CD45 cells have better antifibrotic effect than adipose-derived (AD)-MSCs. In fact, our study revealed that antifibrotic potential of CD45 cells are compromised by the presence of MSCs. This difference was apparently due to significantly high level expressions of matrix metalloproteinases-9 and 13, and the suppression of hepatic stellate cells' (HpSCs) activation in the CD45 cells transplantation group. Mechanism dissection studied in vitro supported the above opposing results and revealed that CD45 cell-secreted FasL induced apoptotic death of activated HpSCs. Further analyses suggest that MSC-secreted transforming growth factor ß and insulin-like growth factor-1 promoted myofibroblastic differentiation of HpSCs and their proliferation. Additionally, the transplantation of CD45 cells led to functional improvement of the liver through repair and regeneration. Thus, BM-derived CD45 cells appear as a superior candidate for the treatment of liver fibrosis due to structural and functional improvement of CCl4 -induced fibrotic liver, which were much lower in case of AD-MSC therapy.

Dopaminergic-primed fetal liver mesenchymal stromal-like cells can reverse parkinsonian symptoms in 6-hydroxydopamine-lesioned mice.

BACKGROUND AIMS: Cell replacement therapy is considered a promising alternative in the treatment of degenerative diseases, and in this context, mesenchymal stromal cells (MSCs) have been proposed for transplantation in Parkinson disease (PD). Thus far, the results of animal studies are found to be inconsistent and inconclusive regarding the therapeutic ability of the cells. This study investigated the efficacy of fetal liver (FL)-MSC-derived dopaminergic (DA) neuronal primed cells for correction of parkinsonian symptoms in mice. METHODS: FL-MSCs were differentiated for 21 days in the presence of a combination of neurotropic factors. The extent of cellular reprogramming was analyzed by quantitative polymerase chain reaction for DA-specific neuronal gene expressions and protein expressions by immuno-cytochemistry. The functionality of the cells was determined by electrophysiology and dopamine release assays. Ten-day-primed neuron-like cells or unprimed MSCs were transplanted into the 6-hydroxydopamine (6-OHDA)-lesioned striatum using a stereotaxic device. Dopamine-secreting properties and behavioral studies were used to assess improvement of parkinsonian symptoms. RESULTS: The differentiated cells expressed DA-specific genes and proteins, while exhibiting a high level of voltage-gated potassium current. Furthermore, neuronal primed cells differentiated into tyrosine hydroxylase immunoreactive and dopamine-secreting functional neuron-like cells. Symptomatic correction of PD in the recipient mice within 2 months of transplantation was also observed. DISCUSSION: FL-MSC-derived primed neuron-like cells integrated into the striatum of PD mice, improving parkinsonian symptoms. This study demonstrates an effective cell-based therapy for PD.

Perspective on liver regeneration by bone marrow-derived stem cells-a scientific realization or a paradox.

Bone marrow (BM)-derived stem cells are reported to have cellular plasticity, which provoked many investigators to use of these cells in the regeneration of nonhematopoietic tissues. However, adult stem cell plasticity contradicts our classic understanding on progressive restriction of the developmental potential of a cell type. Many alternate mechanisms have been proposed to explain this phenomenon; the working hypotheses for elucidating the cellular plasticity of BM-derived stem cells are on the basis of direct differentiation and/or fusion between donor and recipient cells. This review dissects the different outcomes of the investigations on liver regeneration, which were performed with the use of BM-derived stem cells in experimental animals, and reveals some critical factors to explain cellular plasticity. It has been hypothesized that the competent BM-derived stem/progenitor cells, under the influence of liver-regenerating cues, can directly differentiate into hepatic cells. This differentiation takes place as a result of genetic reprogramming, which may be possible in the chemically induced acute liver injury model or at the stage of fetal liver development. Cellular plasticity emerges as an important phenomenon in cell-based therapies for the treatment of many liver diseases in which tissue regeneration is necessary.

Immune response of S. Typhi-derived Vi polysaccharide and outer membrane protein a conjugate in mice.

Typhoid fever is a serious concern precisely in developing nations. Still investigators are exploring a better conjugate partner for Vi-polysaccharide to develop a more effective vaccine for typhoid fever. Here, we cloned and expressed S. Typhi outer membrane protein A (OmpA). The conjugation of Vi-polysaccharide with OmpA was carried out by the carbodiimide (EDAC) method employing ADH as a linker. Total Ig and IgG generated against OmpA, and Vi polysaccharide was quantified by ELISA. Vi polysaccharide alone induced very low levels of Vi polysaccharide antibody. Vi-OmpA conjugate (Vi-conjugate) elicited a robust immune response compared to Vi polysaccharide alone and showed booster response. Further, IgG was only evoked by Vi-OmpA conjugate, not with Vi polysaccharide alone. OmpA antibody induction in both the Vi-OmpA conjugate and OmpA were similar level. Taken together, we show that OmpA as a carrier protein conjugated to Vi polysaccharide is immunogenic. We predict OmpA antibodies will contribute protection along with antibodies generated by Vi-polysaccharide. Past and current literature supports that OmpA is highly conserved protein not only among Salmonellae but entire Enterobacteriacea family with 96-100% identity.

The emerging role of stem cells in ocular neurodegeneration: hype or hope?

Affecting over a hundred million individuals worldwide, retinal diseases are among the leading causes of irreversible visual impairment and blindness. Thus, an appropriate study models, especially animal models, are essential to furthering our understanding of the etiology, pathology, and progression of these diseases. In this review, we provide an overview of retinal disorders in the context of biotherapeutic approaches in these disorders.

Hypoxia improves expansion potential of human cord blood-derived hematopoietic stem cells and marrow repopulation efficiency.

OBJECTIVES: In bone marrow, hematopoietic stem cells (HSCs) reside in the most hypoxic endosteum niche, whereas the proliferating progenitors are located near the relatively oxygen-rich vascular region. High oxygen tension is potentially detrimental to HSCs. The objective of this investigation was to compare cellular, functional, and molecular responses of human umbilical cord blood (UCB)-derived hematopoietic stem and progenitor cells in culture under hypoxic and normoxic conditions. METHODS: CD133-enriched UCB cells were cultured in growth factor containing serum-free and serum-supplemented medium under 5% O(2) (hypoxia) or 21% O(2) (normoxia) for 10 d. The phenotypes of expanded cells were analyzed by flow cytometry and the engraftability by SCID-repopulation assay. The expression of hypoxia-inducible factor (HIF)-1α and some of its target genes was analyzed by real-time RT-PCR. RESULTS: In hypoxic culture, CD34(+) CD38(-) cells were expanded about 27-fold, which was significantly (P < 0.01) higher than that obtained in normoxic culture. Serum-free culture did not support the growth of cells in the presence of 21% O(2) . Myeloid colony-forming potential of cells was significantly (P < 0.05) increased in 5% O(2) compared with 21% O(2) culture. SCID-repopulation efficiency seems to be better preserved in the cells cultured under hypoxic conditions. Hypoxia significantly (P < 0.05) induced the expression of HIF-1α, vascular endothelial growth factor (VEGF), and ABCG2 genes and also upregulated CXCR4 receptor expression. CONCLUSIONS: Low oxygen tension enhanced the proliferation of UCB-derived HSC/progenitor cells and maintenance of SCID-repopulating cells than normoxia. These expanded cells are expected to be beneficial in the patients who lack human leukocyte antigen (HLA)-matched donors.

Bone marrow cells contribute to tubular epithelium regeneration following acute kidney injury induced by mercuric chloride.

BACKGROUND & OBJECTIVES: Acute tubular necrosis (ATN) caused by renal ischaemia, renal hypo-perfusion, or nephrotoxic substances is the most common form of acute kidney injury (AKI). There are a few treatment options for this life-threatening disease and the mortality rate exceeds 50 per cent. In critical cases of AKI the only option is renal transplantation. In the present study we evaluated whether bone marrow cells (BMCs) are involved in regeneration of kidney tubules following acute tubular necrosis in the mouse. METHODS: Six to eight week old C57BL6/J and congenic enhanced green fluorescence protein (eGFP) mice were used. The relative contributions of eGFP-expressing BMCs were compared in two different approaches to kidney regeneration in the mercuric chloride (HgCl 2 )-induced mouse model of AKI: induced engraftment and forced engraftment. In vitro differentiation of lineage-depleted (Lin - ) BMCs into renal epithelial cells was also studied. RESULTS: In the forced engraftment approach, BMCs were found to play a role in the regeneration of tubules of renal cortex and outer medulla regions. About 70 per cent of donor-derived cells expressed megalin. In vitro culture revealed that Lin - BMCs differentiated into megalin, E-cadherin and cytokeratin-19 (CK-19) expressing renal epithelial cells. INTERPRETATION & CONCLUSIONS: The present results demonstrate that Lin - BMCs may contribute in the regeneration of renal tubular epithelium of HgCl 2 -induced AKI. This study may also suggest a potential role of BMCs in treating AKI.

Prognostic significance of D-dimer, natural anticoagulants and routine coagulation parameters in acute pancreatitis.

AIM: This study was conducted to assess the predictive value of coagulation abnormalities in determining disease severity and prognosis of acute pancreatitis (AP). METHODS: Patients of AP and 25 healthy volunteers were included in this prospective observational study. The final outcomes were disease severity assessed by Computed Tomography Severity Index, Acute Physiological Assessment and Chronic Health Evaluation--II, presence of organ failure and mortality. Prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT), fibrinogen, antithrombin-III (AT-III), protein-C, and protein-S levels were assessed on day 0, 3 and 7 of admission. RESULTS: Of the 38 patients included, 13 died. Mean PT and TT were similar between patients and controls on any given day. PTT showed elevation on day 3 and 7 (p = 0.001) compared to controls, although fibrinogen and D-dimer were significantly higher in patients on all days. Protein C and AT-III were significantly lower in patients and more so in non survivors ( (p = 0.001)) than controls. Multiple logistic regression analysis revealed D-dimer levels > or = 400 - 800 ng/ml and AT- III level of < 71% at admission were associated with high mortality (OR 11.2, AUROC 0.70 and OR 16.6, AUROC 0.82 respectively) as well as predicted organ failure. CONCLUSION: Serum D-dimer and antithrombin-III levels can be used to assess disease severity and predict outcome of patients with acute pancreatitis.

Hepatocyte nuclear factor-4alpha induces transdifferentiation of hematopoietic cells into hepatocytes.

Hematopoietic stem cells can directly transdifferentiate into hepatocytes because of cellular plasticity, but the molecular basis of transdifferentiation is not known. Here, we show the molecular basis using lineage-depleted oncostatin M receptor beta-expressing (Lin(-)OSMRbeta(+)) mouse bone marrow cells in a hepatic differentiation culture system. Differentiation of the cells was marked by the expression of albumin. Hepatocyte nuclear factor (HNF)-4alpha was expressed and translocated into the nuclei of the differentiating cells. Suppression of its activation in OSM-neutralized culture medium inhibited cellular differentiation. Ectopic expression of full-length HNF4alpha in 32D myeloid cells resulted in decreased myeloid colony-forming potential and increased expression of hepatocyte-specific genes and proteins. Nevertheless, the neohepatocytes produced in culture expressed active P450 enzyme. The obligatory role of HNF4alpha in hepatic differentiation was confirmed by transfecting Lin(-)OSMRbeta(+) cells with dominant negative HNF4alpha in the differentiation culture because its expression inhibited the transcription of the albumin and tyrosine aminotransferase genes. The loss and gain of functional activities strongly suggested that HNF4alpha plays a central role in the transdifferentiation process. For the first time, this report demonstrates the mechanism of transdifferentiation of hematopoietic cells into hepatocytes, in which HNF4alpha serves as a molecular switch.

The therapeutic effect of bone marrow-derived liver cells in the phenotypic correction of murine hemophilia A.

The transdifferentiation of bone marrow cells (BMCs) into hepatocytes has created enormous interest in applying this process to the development of cellular medicine for degenerative and genetic diseases. Because the liver is the primary site of factor VIII (FVIII) synthesis, we hypothesized that the partial replacement of mutated liver cells by healthy cells in hemophilia A mice could manage the severity of the bleeding disorder. We perturbed the host liver with acetaminophen to facilitate the engraftment and hepatic differentiation of lineage-depleted enhanced green fluorescent protein-expressing BMCs. Immunohistochemistry experiments with the liver tissue showed that the donor-derived cells expressed the markers of both hepatocytes (albumin and cytokeratin-18) and endothelial cells (von Willebrand factor). The results of fluorescent in situ hybridization and immunocytochemistry experiments suggested that differentiation was direct in this model. The BMC-recipient mice expressed FVIII protein and survived in a tail clip challenge experiment. Furthermore, a coagulation assay confirmed that the plasma FVIII activity was maintained at 20.4% (+/- 3.6%) of normal pooled plasma activity for more than a year without forming its inhibitor. Overall, this report demonstrated that BMCs rescued the bleeding phenotype in hemophilia A mice, suggesting a potential therapy for this and other related disorders.

PDGFRα mediated survival of myofibroblasts inhibit satellite cell proliferation during aberrant regeneration of lacerated skeletal muscle.

Aberrant regeneration or fibrosis in muscle is the denouement of deregulated cellular and molecular events that alter original tissue architecture due to accumulation of excessive extracellular matrix. The severity of the insult to the skeletal muscle determines the nature of regeneration. Numerous attempts at deciphering the mechanism underlying fibrosis and the subsequent strategies of drug therapies have yielded temporary solutions. Our intent is to understand the interaction between the myofibroblasts (MFs) and the satellite cells (SCs), during skeletal muscle regeneration. We hypothesize that MFs contribute to the impairment of SCs function by exhibiting an antagonistic influence on their proliferation. A modified laceration based skeletal muscle injury model in mouse was utilized to evaluate the dynamics between the SCs and MFs during wound healing. We show that the decline in MFs' number through inhibition of PDGFRα signaling consequently promotes proliferation of the SCs and exhibits improved skeletal muscle remodeling. We further conclude that in situ administration of PDGFRα inhibitor prior to onset of fibrosis may attenuate aberrant regeneration. This opens new possibility for the early treatment of muscle fibrosis by specific targeting of MFs rather than transplantation of SCs.

A Two-Stage Process for Differentiation of Wharton's Jelly-Derived Mesenchymal Stem Cells into Neuronal-like Cells.

With no permanent cure for neurodegenerative diseases, the symptoms reappear shortly after the withdrawal of medicines. A better treatment outcome can be expected if the damaged neurons are partly replaced by functional neurons and/or they are repaired using trophic factors. In this regard, safe cell therapy has been considered as a potential alternative to conventional treatment. Here, we have described a two-stage culture process to differentiate Wharton Jelly mesenchymal stem cells (WJ-MSCs) into neuronal-like cells in the presence of various cues involved in neurogenesis. The fate of cells at the end of each stage was analyzed at the morphometric, transcriptional, and translational levels. In the first stage of priming, constitutively, wingless-activated WJ-MSCs crossed the lineage boundary in favor of neuroectodermal lineage, identified by the loss of mesenchymal genes with concomitant expression of neuron-specific markers, like SOX1, PAX6, NTRK1, and NEUROD2. Neuronal-like cells formed in the second stage expressed many mature neuronal proteins like Map2, neurofilament, and Tuj1 and possessed axon hillock-like structures. In conclusion, the differentiation of a large number of neuronal-like cells from nontumorigenic and trophic factors secreting WJ-MSCs promises the development of a therapeutic strategy to treat neurodegenerative diseases.

Alteration of Mitochondrial Function in Oxidative Stress in Parkinsonian Neurodegeneration: A Cross-Sectional Study.

CONTEXT: Appropriate mitochondrial function and oxidative balance are critical to neuronal survival. Accumulation of reactive oxygen species leads to oxidative stress that can cause free radical damage to biomolecules of the cell components and the molecules in the cellular milieu that eventually lead to a variety of chronic diseases including neurodegenerative disorders. Mitochondrial dysfunction initiates neuronal apoptosis thereby leading to neurodegenerative diseases including Parkinson's disease (PD). AIM: To evaluate oxidative stress vis-a-vis mitochondrial function (Cytochrome C oxidase activity) in PD patients, Parkinson plus syndrome (PPS) patients in comparison with healthy controls (HCs). SETTINGS AND DESIGN: Cross-sectional Study. METHODS: We assessed oxidative stress by chemiluminescence using luminol, and cytochrome c oxidase activity (CCO) by CCO kit using spectrophotometry in PD patients (n = 80), PPS patients (n = 40), and HCs (n = 40). STATISTICAL ANALYSIS: Data were presented as number (%) or mean ± SD/median as approximate. Quantitative baseline variables were compared among the groups using one-way ANOVA and qualitative variables were compared using Chi-square test. The difference in median was compared using Kruskal-Wallis test followed by Post-hoc Bonferronni correction. RESULTS: Compared to HCs (Median 7.53 ± 15.58 RLU/sec/cell), ROS level in PD (14.13 ± 29.5), and PPS (17.43 ± 15.91) patients was significantly higher (P = 0.0029: HC vs, PD & P = 0.0500: HC vs. PPS). Also, ROS in PD patients (14.13 ± 29.5) was higher that PPS patients (17. 43 ± 15.91) but the difference was not statistically significant (P = 0.84). The CCO activity was found to be diminished in PD (Median: 0.025 ± 0.013 units/ml) and PPS patients (0.027 ± 0.008) in comparison to HCs (0.117 ± 0.049). CONCLUSION: Mitochondrial dysfunction and oxidative stress is associated with PD and PPS and may play an important role in etiopathogenesis. Though the cause-effect conundrum has not been comprehensively probed but addressing oxidative stress and mitochondrial damage may serve as an adjunctive therapy for PD and PPS. Iron metabolism as reflected in the red cell indices may aid in differentiating PD from PPS.

IL-6-174 G/C and ApoE gene polymorphisms in Alzheimer's and vascular dementia patients attending the cognitive disorder clinic of the All India Institute of Medical Sciences, New Delhi.

BACKGROUND: Remarkable improvement in the life expectancy of the Indian population is expected to commensurate with the increase in number of dementia cases. Among various types of dementia, Alzheimer's disease (AD) and vascular dementia (VaD) are common and widely studied. We evaluated the role of apolipoprotein E (ApoE) and interleukin-6 (IL-6)-174 G/C gene polymorphism along with serum IL-6 levels in AD and VaD patients. METHODS: The polymorphisms in ApoE and IL-6-174 G/C genes were assessed using RFLP. Serum IL-6 level was measured by ELISA. RESULTS: The allele ε4 of the ApoE gene was found to be associated with AD and VaD patients (p < 0.05). No association of IL-6-174 G/C polymorphism was observed in AD patients, while the IL-6-174 C allele increased the odds of having VaD twofold. Regression analysis to assess possible interaction between ApoE and the IL-6-174 G/C genes revealed that presence of both the ε4 and C alleles increased the odds of having AD 13.75-fold and VaD 14.7-fold. Serum IL-6 levels did not correlate with either presence or severity of disease among AD or VaD patients. CONCLUSION: The ApoE ε4 allele is an important genetic marker for AD and VaD. Presence of both ApoE ε4 and IL-6 C genes increases the OR of having AD and VaD markedly.

Hematopoietic progenitors from early murine fetal liver possess hepatic differentiation potential.

Bipotential hepatoblasts differentiate into hepatocytes and cholangiocytes during liver development. It is believed that hepatoblasts originate from endodermal tissue. Here, we provide evidence for the presence of hepatic progenitor cells in the hematopoietic compartment at an early stage of liver development. Flow cytometric analysis showed that at early stages of liver development, approximately 13% of CD45(+) cells express Delta-like protein-1, a marker of hepatoblasts. Furthermore, reverse transcriptase-PCR data suggest that many hepatic genes are expressed in these cells. Cell culture experiments confirmed the hepatic differentiation potential of these cells with the loss of the CD45 marker. We observed that both hematopoietic activity in Delta-like protein-1(+) cells and hepatic activity in CD45(+) cells were high at embryonic day 10.5 and declined thereafter. Clonal analysis revealed that the hematopoietic fraction of fetal liver cells at embryonic day 10.5 gave rise to both hepatic and hematopoietic colonies. The above results suggest a common source of these two functionally distinct cell lineages. In utero transplantation experiments confirmed these results, as green fluorescent protein-expressing CD45(+) cells at the same stage of development yielded functional hepatocytes and hematopoietic reconstitution. Since these cells were unable to differentiate into cytokeratin-19-expressing cholangiocytes, we distinguished them from hepatoblasts. This preliminary study provides hope to correct many liver diseases during prenatal development via transplantation of fetal liver hematopoietic cells.

Evaluation of four methods for processing human cord blood and subsequent study of the expansion of progenitor stem cells isolated using the best method.

BACKGROUND AIMS: The application of cord blood (CB)-derived hematopoietic cells in allogeneic transplantation has been restricted because of the unavailability of adequate cell numbers from one unit of CB and their delayed engraftment in recipients. The main purpose of this study was to develop an economic process for isolating nucleated cells from CB and expanding enriched CD133(+) cells. METHODS: Four protocols for processing CB, using different combinations of density-gradient centrifugation, hydroxyethyl starch (HES) and NH4Cl treatment, were compared regarding the yields of CD45(+), CD34(+)/CD133(+) and colony-forming cells. CD133-enriched cells were expanded for 10 days in the presence of hematopoietic growth factor-supplemented medium. The performance of the culture was evaluated by analyzing colony-forming potential, fold-expansion of primitive hematopoietic stem cells (HSC) and lineage commitment by flow cytometry. RESULTS: A two-step treatment of CB with HES followed by NH4Cl resulted in the highest recovery of CD45(+) (84.3%) and CD34(+) (69.1%) cells compared with that present in umbilical CB. A suspension culture of CD133-enriched cells resulted in an average 150-fold increase in nucleated cells, of which CD133(+), CD34(+) and CD34(+) CD38 cell expansions were 17.2+/-1.3, 40+/-4.6, and 6.9+/-1.1 fold, respectively. The total myeloid colony-forming cell count was almost 3800-fold higher than that present in the processed CB. CONCLUSIONS: The highest yields of nucleated and progenitor stem cells were obtained with a two-step processing of CB. The CD133(+) cells obtained by this method are expected to yield enough hematopoietic progenitors for potential allogeneic transplantation.

Volume, conductivity, and scatter parameters of leukocytes as early markers of sepsis and treatment response.

INTRODUCTION: Morphologic changes in the size and granularity of leukocytes seen in sepsis could be measured using the volume, conductivity, and scatter (VCS parameters) from the automated hematology analyzers. The objective of this study is to find the clinical usefulness of VCS parameters as possible indicators of sepsis and to determine the effect of treatment on these parameters. METHODS: This observational study was conducted in a tertiary level hospital in India. Hemogram and VCS parameters obtained from LH 750 (Beckman coulter, Fullerton, CA) from 134 proven blood culture-positive cases of sepsis were reviewed on the day of culture positivity (day 0), day 3, and day 7 were analyzed and compared with those of samples from otherwise healthy 100 participants. Statistical analysis of data was done, and cutoff value was established using receiver-operator characteristic curve. RESULTS: Out of 134 culture-positive cases, 55.2% (n = 74) Gram-negative and 44.8% (n = 60) Gram-positive bacteria were isolated. The mean neutrophil volume (MNV) and mean monocyte volume (MMV) were higher in the sepsis group compared to that of the control group (165.43 ± 18.21 vs. 140.59 ± 7.6, P = 0.001 for MNV and 179.8 ± 14.16 vs. 164.54 ± 9.6, P = 0.001 for MMV). A significant decrease in MNV and MMV was observed with the initiation of the treatment. Significant changes in scatter and conductivity parameters were also noticed. A cutoff value of 150.2 for MNV gave a sensitivity and specificity of 79.1% and 95%, respectively, with an area under the curve (AUC) of 92.3%. With a cutoff of 168.3, MMV had a sensitivity of 80.6% and specificity of 77.5%, AUC of 83%. CONCLUSION: VCS parameters such as MNV and MMV can be easily obtained by an automated hematology analyzer and could be used for early detection and therapeutic response in sepsis.

Serum S100B levels in patients with depression.

BACKGROUND: The biochemical basis of depression has been related to blood-brain barrier (BBB) allowing/restricting a number of components to enter the brain milieu from the peripheral plasma milieu. S100B has been associated with BBB damage and is used as a marker of its integrity. Several studies have reported that depressive patients have increased levels of S100B in serum and cerebrospinal fluid. MATERIALS AND METHODS: Forty-two confirmed cases of depression, 13-25 years of ages were recruited from the Department of Psychiatry, All India Institute of Medical Sciences during the period from January 2013 to June 2014 along with 42 healthy controls of comparable age and sex. Psychometric evaluation of the patients and controls was done to assess the severity of depression using Beck's Depression Inventory-II and Hamilton Depression Rating Scale. Medical assessment and laboratory investigations were done. Serum S100B levels were measured using Sandwich ELISA. The results obtained were statistically analyzed. RESULTS: Levels of serum S100B were significantly elevated in patients with major depression as compared to controls. Significantly higher levels of S100B were seen only in females as compared to their healthy counterparts. Serum S100B was higher in depressed participants with the recurrent disorder than those with single episode. No correlation of levels of this marker was seen with clinical severity of the patients. It was found that with increased duration of illness for which the patient was being treated with antidepressants, the patients had higher levels of S100B. CONCLUSIONS: Serum S100B can be used as a biomarker of depression.

Monitoring Natural Killer Cell Function in Human Ovarian Cancer Cells of Ascitic Fluid.

Natural killer (NK) cells are the major effectors of the innate immune system when activated resulting in modulation of immune response of the host defense through target cell lysis and secretion of cytokines. Precise functions of NK cells are essential for the treatment outcome of different virus infections and malignant diseases. NK cells impart cytotoxic effect to the target cells lacking MHC class I molecules and thus the final readout of the activity is death of target cells. The NK cell function is evaluated by the 51Cr-release and/or flow cytometry-based assays. In the present protocol, we have determined the activation of NK cells by the liberation of IL-10 and IFNγ, and subsequently its function by enumerating the number of dead tumor cells originally isolated from the ascitic fluid of ovarian cancer patients. The entire assay is based on cells of the healthy donors and patients. Besides determining function, this method is able to demarcate between NK-cell sensitive and insensitive tumor cells. This technique enables researchers to study NK cell functions in healthy donors or in patients to reveal their impact on different malignancies and to further discover new therapeutic strategies.