Partial purification and characterization of a novel human factor that augments the expression of class I MHC antigens on tumour cells

A cytokine which augments the expression of major histocompatibility complex (MHC) I antigens on K562 and gastric carcinoma tumour (HR) cells, has been isolated from the culture supernatant of Concanavalin-A (Con-A) activated human peripheral blood mononuclear cells. The factor, termed MHC augmenting factor (MHC- AF) has been partially purified by Sephadex G- 100 column chromatography, preparative isoelectric focusing and HPLC with ion- exchange as well as sizing columns. MHC-AF activity is associated with a 35 kDa molecule which has pI of 6.0. Interferon (IFN)-α, \, tumour necrosis factor (TNF), Interleukin (IL)-2, IL-4, IL-5 and IL-7 had no significant effect in MHC- AF bioassay, but IFN-γ had significant MHC-AF activity. Antibodies to IFN-α, IFN-\ and TNF-α did not block the activity of MHC-AF, but anti-IFN-y antibodies could partially neutralize the activity. However, unlike IFN-γ, MHC-AF activity was resistant to pH 2.0 treatment. Purified MHC-AF preparations did not have any activity in WISH cell/encephalo myocarditis virus (EMC) IFN bioassays. In addition, anti-IFN-y affinity column did not retain MHC-AF activity. These results indicate that a MHC-AF distinct from IFN-γ, is produced by activated human mononuclear cells.     

Purification and characterization of two major lectins fromVigna mungo (blackgram)

Blackgram (Vigna mungo L. Hepper)seeds contain two galactose-specific lectins, BGL-I and BGL-II. BGL-I was partially purified into two monomeric lectins which were designated as BGL-I-1 (94 kDa) and BGL-I-2 (89 kDa). BGL-II is a monomeric lectin of 83 kDA. The purified lectins were associated with galactosidase activities. BGL-I-1 and BGL-II were copurified with α-galactosidase activity while BGL-I-2 was largely associated with β-galactosidase activity. These lectins agglutinate trypsin treated rabbit erythrocytes, but not the human erythrocytes of A, B or O groups. They were stable between pH 3·5 and 7·5 for their agglutination. The lectins did not show any metalion requirement. They were inactivated at 50°C. The lectin activity was inhibited by D-galactose (0·1 mM). The Scatchard plots of galactose binding to these lectins are nonlinear and biphasic curves indicative of multiple binding sites. The data show that the monomeric lectins have both lectin and galactosidase activities suggestive of a bifunctional protein.     

Ethyl methanesulphonate induced changes in the differentiation, structure and functions of spermatozoa of the house rat,Rattus rattus L.

Intraperitoneal administration of 500 mg/kg and 625 mg/kg doses of the germ cell mutagen, ethyl methanesulphonate (EMS) in 5 consecutive days to the house rat,Rattus rattus caused a dose-dependent reduction in its body weight, cauda epididymides weight, concentration, motility and percentage of live spermatozoa with simultaneous increase in the percentage of their abnormal forms. Compared to 0·65% spermatozoa with abnormal heads in the cauda epididymidis of untreated control rats, 24·86% and 65·72% such spermatozoa were observed in rats on day 14 post treatment with 500 mg/kg and 625 mg/kg doses of EMS respectively. On day 28 post treatment corresponding values for abnormal spermatozoa were 16·21% and 14·32%. Similarly, spermatozoa with abnormal flagella increased from 0.78% in control rats to 9·25% and 5·75% on day 14 post treatment of 500 and 625 mg/kg doses of EMS respectively and declined to 2·91% and 2·40% on day 28 post treatment. Abnormality in the sperm head was mainly due to acrosomelessness and in the flagellum due to bending at proximal region. However, the main effect of EMS was the development of spermatozoa without or deformed acrosomes which may impair the fertility of rats. Analysis of various stages of differentiation of spermatozoa inthe testis revealed that population of preleptotene and pachytene spermatocytes and of round spermatids showed a gradual decline which became significantly less than controls on day 28 of EMS treatment. Occurrence of abnormal heads of testicular spermatids indicated that the sperm head abnormalities originated in the testis during late spermiogenesis.     

Pre-conditioning mesenchymal stromal cell spheroids for immunomodulatory paracrine factor secretion

Background aimsMesenchymal stromal cells (MSCs) exhibit the inherent potential to regulate multiple signaling pathways and cell types that contribute to the pathogenesis of inflammatory and immune diseases. However, more recent studies have suggested that the secretion of immunomodulatory factors by MSCs can be enhanced by three-dimensional aggregation or pro-inflammatory cytokine treatment.MethodsHuman MSC spheroids were formed by forced aggregation into agarose micro-wells and subsequently cultured in either minimal essential medium alpha supplemented with fetal bovine serum or serum-free, defined MesenCult-XF medium (STEMCELL Technologies, Vancouver, Canada). A subset of the spheroids were treated with pro-inflammatory cytokines interferon (IFN)-γ or tumor necrosis factor (TNF)-α or both for 4 days. Immunomodulatory factor (prostaglandin E₂, indoleamine 2,3-dioxygenase, transforming growth factor-β1 and interleukin-6) secretion was quantified after 4 days of culture, and the immunomodulatory activity of MSCs was assessed by quantifying activated macrophage expression of TNF-α after trans-well co-culture.ResultsCulturing human MSCs as three-dimensional aggregates increased secretion of immunomodulatory paracrine factors, which was enhanced further by treatment with IFN-γ and TNF-α, demonstrating that these parameters can synergistically enhance endogenous human MSC immunomodulatory properties. However, immunomodulatory factor secretion was found to be highly dependent on the composition of cell culture medium. Human MSCs cultured in MesenCult-XF medium displayed significantly less expression of prostaglandin E₂, indoleamine 2,3-dioxygenase, transforming growth factor-β1 and interleukin-6 compared with human MSCs cultured in medium supplemented with fetal bovine serum. Finally, pre-conditioning of human MSC spheroids with IFN-γ and TNF-α resulted in greater immunomodulatory activity in a macrophage co-culture assay.ConclusionsAltogether, engineering the environment of human MSCs to develop pre-conditioning strategies for enhancing human MSC immunomodulation may be a simple approach for improving MSC-based therapies for the treatment of inflammatory and immune diseases.     

Biochemical and immunofluorescence analysis of the constitutively expressed HSP27 stress protein in monkey CV-1 cells

The α-crystallin-related stress protein HSP27, which promotes cellular resistance to different types of stress, is constitutively expressed during the growth of several primate tissue culture cells. Here, we report an analysis of the cellular localization of this protein in CV-1 monkey cells. Following cell lysis and fractionation in the absence of detergent about 2 5 % of the cellular content of HSP27 was recovered in the particu late fractions while the remaining of this protein was in the soluble cytoplasmic fraction. This association of HSP27 with particulate fractions was no more observed when cells were lysed in the presence of non-ionic detergent or when cells were pretreated with drugs, such as monensin and colcemid, that disrupt cytoskeletal architecture. Immunofluorescence analysis revealed that HSP27 is concentrated in a polarized perinuclear zone of CV-1 cells from where microtubules radiate. The particular locale of HSP27 was investigated in cells exposed to drugs or treatments, such as monensin, colcemid, cold stess and serum starvation, that disrupt the cellular architecture of microtubules. A correlation was observed between HSP27 cellular locale and microtubules integrity. Our results suggest a possible interaction of a fraction of HSP27 with cytoplasmic organelles or structures, different from the Golgi apparatus, whose distribution depends upon the organization of microtubules.     

Flowcytometric assessment of intracellular interferon -γ production in human CD4 + ve T-cells on mycobacterial antigen stimulation

Interferon-(IFN-γ) has been considered to be a critical protective immunomodulatory component against Mycobacterium tuberculosis (M. tb.) infection. In this study T-cell proliferation and IFN-γ production upon stimulation with M. tb. were assessed in patients of pulmonary tuberculosis and healthy contacts. The studies were based on lymphocyte transformation test and detection of intracellular IFN-γ production by CD4 + ve T-cells by flowcytometry. Patients showed lower levels of proliferation, the stimulation index being in the range of 2.17 1.1 (mean + SD) compared to the contacts (SI = 4 59±1.6) (P < 0.01). The kinetics of intracellular induction of IFN-γ on M. tb. stimulation showed a proportional increase in the CD4 + ve T-cell population. The increase was maximal between 96–120 h of culture. In healthy contacts the number of IFN-γ expressing CD + ve T-cells increased to 2.5 to 41 × 10⁴ cells/ml in M. tb. stimulated cultures compared to control cultures (0.1 – 15 × 10⁴). In contrast patients showed no/marginal increase in CD4 + ve T-cell population expressing intracellular IFN-γ Thus the lack of induction of IFN in CD4 + ve T-cells in patients could be a critical shortcoming in their ability to combat tubercle bacilli infection.     

Plant regeneration from protoplasts ofCapsicum annuum L. cv. California Wonder

Plant regeneration from mesophyll protoplasts of pepper,Capsicum annuum L. cv. California Wonder has been demonstrated via shoot organogenesis. Protoplasts isolated from fully expanded leaves of 3-week-old axenic shoots when cultured in TM medium supplemented with 1 mg l ⁻¹ NAA, 1 mg l ⁻¹ 2,4-D, 0 5 mg l ⁻¹ BAP (CM 1) resulted in divisions with a frequency ranging from 20–25 %. Antioxidant ascorbic acid and polyvinylpyrrolidone (PVP) in the medium and incubation in the dark helped overcome browning of protoplasts. Microcalli and macrocalli were formed in TM medium containing 2 mg l ⁻¹ NAA and 0.5 mg l ⁻¹ BAP (CM II) and MS gelled medium containing 2 mg 1 ⁻¹ NAA and 0 5 mg 1 ⁻¹ BAP (CM III), respectively. Regeneration of plantlets was possible via caulogenesis. Microshoots, 2–5 percallus appeared on MS gelled medium enriched with 0.5 mg l ⁻¹ IAA, 2mg l ⁻¹ GA and l0mg l ⁻¹ BAP (CM IVc). Rooting of microshoots was obtained on half strength gelled medium containing 1 mg l ⁻¹ NAA and 0.5mg l ⁻¹ BAP. Protoplasts isolated from cotyledons failed to divide and degenerated eventually.     

Efforts in diagnosing early leprosy using serological techniques

Skin scrapings obtained from the lesions of leprosy patients of all types showed 96 % positivity to the serum antibody competition test using monoclonal antibody (ML04)to 35 kDa antigen of Mycobacterium leprae. Further, in vitro culture of full thickness skin biopsies from lepromatous patients were noted to release IgG antibodies toM. leprae with a peak antibody response at 48 h. The significance of this local antibody response toM. leprae in skin has been discussed for its possible use in diagnosing early leprosy.     

Micropropagation of selected somaclones ofBegonia andSaintpaulia

Begonia x elatior plantlets which regenerated from leaf disk callus showed variations in plant morphology, number of flowers per plant, and flower size. Variations in flowering period, number of flowers per plant, and flower morphology were observed in Saintpaulia ionantha L. plants directly regenerated from leaf disk explants. The cytokinins, benzylaminopurine and zeatin, tested in the culture medium did not affect the basic plant characteristics including flower colour which remained stable in both species. Micropropagation of selected somaclones having the desirable trait of high number of flowers per plant was stable in the MV2 and MV3 generations.     

Missing self by heterogeneous natural killer cells

Some murine (YAC, P815 and SP20) and human (Molt4, Raji and HR7) tumour cell lines were (i) treated with IFN-γ for inducing enhanced expression of MHC class I antigen, or (ii) given a brief treatment with citrate buffer (pH 3.0), which resulted in denaturation of class I MHC antigens on these tumour cells. IFL-γ or acid treated tumour cells were used as unlabelled competing targets in cold target inhibition assays. The results indicated that the competing ability of acid-treated tumour cells remained unaltered, whereas IFN-γ treated tumour cells competed with significantly less efficiency. These results have been evaluated in light of the current view of NK cell development and the expression of inhibitory receptors for MHC class I molecules (IRMs), on NK cells. A modified view on NK cell heterogeneity based upon IRM expression has been proposed which reconciles several apparently discordant observations about the activity and role of NK cells. Two classes of NK cells have been proposed. Type I NK ceils have target recognition receptors which do not recognize autologous normal cells, lack IRMs, and may participate in first line of defence against transformed cells in vivo. Type II NK cells have target recognition receptors for autologous normal cells and express at least one self-reactive IRM in order to prevent auto-killing. Type II NK cells participate in killing those transformed cells which down-regulate their MHC class I expression in order to escape cytotoxic T-cell surveillance. It is also postulated that mechanism of inverse correlation of target cell MHC class I expression levels and their susceptibility to NK cells, involves interference model of missing self hypothesis for type I NK cells and inhibitory signal model of missing self hypothesis for type II NE cells. Finally, it is proposed that acid treatment of tumour cells enhances their lysis susceptibility by making them additionally susceptible to type II NK cells, rather than enhancing their killing by type I NK cells. This proposition would explain the lack of effect of acid treatment on the competing ability of tumour cells, when target cells are only lysed by type I NE cells.     

Characteristics of arachidonic acid metabolism of human endothelial cells in culture

Human umbilical endothelial cells in culture retain differentiated morphological and functional characterization in primary culture and even in the early subcultures, after which they begin to degenerate. We have studied the morphological and biochemical characterization (ability to produce prostacyclin, prostaglandin E₂ and thromboxane A₂ in culture) of endothelial cells in the first seven subcultures. In addition the influence of serum and endothelial cell growth factor added to the culture medium have been evaluated. With 20% normal human serum, cell proliferation is faster than with the same concentration of human fetal or bovine fetal serum.After the 3rd passage, morphological and growth alterations become observable in the endothelial cells. However, prostacyclin, prostaglandin E₂ and thromboxane A₂ production showed no variations during the study.     

Bioreactor expansion of human neural precursor cells in serum‐free media retains neurogenic potential

Human neural precursor cells (hNPCs), harvested from somatic tissue and grown in vitro, may serve as a source of cells for cell replacement strategies aimed at treating neurodegenerative disorders such as Parkinson's disease (PD), Huntington's disease (HD), and intractable spinal cord pain. A crucial element in a robust clinical production method for hNPCs is a serum‐free growth medium that can support the rapid expansion of cells while retaining their multipotency. Here, we report the development of a cell growth medium (PPRF‐h2) for the expansion of hNPCs, achieving an overall cell‐fold expansion of 10¹³ over a period of 140 days in stationary culture which is significantly greater than other literature results. More importantly, hNPC expansion could be scaled‐up from stationary culture to suspension bioreactors using this medium. Serial subculturing of the cells in suspension bioreactors resulted in an overall cell‐fold expansion of 7.8 × 10¹³ after 140 days. These expanded cells maintained their multipotency including the capacity to generate large numbers of neurons (about 60%). In view of our previous studies regarding successful transplantation of the bioreactor‐expanded hNPCs in animal models of neurological disorders, these results have demonstrated that PPRF‐h2 (containing dehydroepiandrosterone, basic fibroblast growth factor and human leukemia inhibitory factor) can successfully facilitate the production of large quantities of hNPCs with potential to be used in the treatment of neurodegenerative disorders. Biotechnol. Bioeng. 2010. 105: 823–833. © 2009 Wiley Periodicals, Inc.     

Impact of bone marrow-derived mesenchymal stromal cells on experimental xenogeneic graft-versus-host disease

Background aimsGraft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous studies have suggested that mesenchymal stromal cells (MSCs) could exert potent immunosuppressive effects.MethodsThe ability of human bone marrow derived MSCs to prevent xenogeneic GVHD in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and in NOD/SCID/interleukin-2Rγ(null) (NSG) mice transplanted with human peripheral blood mononuclear cells (PBMCs) was assessed.ResultsInjection of 200 × 10⁶ human PBMCs intraperitoneally (IP) into sub-lethally (3.0 Gy) irradiated NOD/SCID mice also given anti-asialo GM₁ antibodies IP 1 day prior and 8 days after transplantation induced lethal xenogeneic GVHD in all tested mice. Co-injection of 2 × 10⁶ MSCs IP on day 0 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Similarly, injection of 30 × 10⁶ human PBMCs IP into sub-lethally (2.5 Gy) irradiated NSG mice induced a lethal xenogeneic GVHD in all tested mice. Injection of 3 × 10⁶ MSCs IP on days 0, 7, 14 and 21 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs.ConclusionsInjection of MSCs did not prevent xenogeneic GVHD in these two humanized mice models.     

Human mesenchymal stromal cells transiently increase cytokine production by activated T cells before suppressing T-cell proliferation: effect of interferon-γ and tumor necrosis factor-α stimulation

Background aimsMesenchymal stromal cells (MSCs) suppress T-cell proliferation, especially after activation with inflammatory cytokines. We compared the dynamic action of unprimed and interferon (IFN)-γ plus tumor necrosis factor (TNF)-α–pretreated human bone marrow–derived MSCs on resting or activated T cells.MethodsMSCs were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) at high MSC-to-PBMC ratios in the absence or presence of concomitant CD3/CD28-induced T-cell activation. The kinetic effects of MSCs on cytokine production and T-cell proliferation, cell cycle and apoptosis were assessed.ResultsUnprimed MSCs increased the early production of IFN-γ and interleukin (IL)-2 by CD3/CD28-activated PBMCs before suppressing T-cell proliferation. In non-activated PBMC co-cultures, low levels of IL-2 and IL-10 synthesis were observed with MSCs in addition to low levels of CD69 expression by T cells and no T-cell proliferation. MSCs also decreased apoptosis in resting and activated T cells and inhibited the transition of these cells into the sub-G0/G1 and the S phases. With inhibition of indoleamine 2,3 dioxygenase, MSCs increased CD3/CD28-induced T-cell proliferation. After priming with IFN-γ plus TNF-α, MSCs were less potent at increasing cytokine production by CD3/CD28-activated PBMCs and more effective at inhibiting T-cell proliferation but had preserved anti-apoptotic functions.ConclusionsUnprimed MSCs induce a transient increase in IFN-γ and IL-2 synthesis by activated T cells. Pre-treatment of MSCs with IFN-γ plus TNF-α may increase their effectiveness and safety in vivo.     

Association of HSP90 with the heme-regulated eukaryotic initiation factor 2α kinase—A collaboration for regulating protein synthesis

Among the various heat shock proteins (HSPs), members of the HSP70 and HSP90 families have drawn particular attention due to their heat shock-unrelated functions. HSP90, an ubiquitous and abundant member of the HSP90 family has been shown to be associated with a large array of protein factors. These proteins reside in the nucleus as well as in the cytoplasm and are involved in various physiological processes, such as, regulation of chromatin structure, cell cycle, cytoskelelal architecture, protein trafficking and protein synthesis. In this article, we focus our interest on the role of HSP90 in protein synthesis. Recent data obtained from a few laboratories strongly suggest that HSP90 interacts with the heme-regulated eukaryotic initiation factor 2α (elF-2α) kinase, also called the heme-regulated inhibitor, and causes its activation which leads to inhibition of protein synthesis. On the basis of data reported from various laboratories, including our own, we propose a possible model on the mechanism of HSP90-mediated activation of heme-regulated inhibitor and regulation of protein synthesis.     

Scientific publications of DW Wittekind

Abstract

Loss of cells in the human disc due to programmed cell death (apoptosis) is a major factor in the aging and degenerating human intervertebral disc. Our objective here was to determine if thymosin β₄ (TB4), a small, multifunctional 5kDa protein with diverse activities, might block apoptosis in human annulus cells cultured in monolayer or three-dimensional (3D) culture. Apoptosis was induced in vitro using hydrogen peroxide or serum starvation. Annulus cells were processed for identification of apoptotic cells using the TUNEL method. The percentage of apoptotic cells was determined by cell counts. Annulus cells also were treated with TB4 for determination of proliferation, and proteoglycan production was assessed using cell titer and 1,2 dimethylmethylamine (DMB) assays and histological staining. A significant reduction in disc cell apoptosis occurred after TB4 treatment. The percentage of cells undergoing apoptosis decreased significantly in TB4 treated cells in both apoptosis induction designs. TB4 exposure did not alter proteoglycan production as assessed by either DMB measurement or histological staining. Our results indicate the need for further studies of the anti-apoptotic effect of TB4 and suggest that TB4 may have therapeutic application in future biological therapies for disc degeneration.     

Liver stem cells: a two compartment system

Hepatocytes and biliary epithelia are phenotypically very dissimilar, but share a common ancestry. Hepatocytes regenerate very efficiently, and their division potential indicates that many of them are functional stem cells. When hepatocyte-damaging agents also impair the regenerative ability of surviving hepatocytes, a potential stem cell system of biliary origin is activated to generate new hepatocytes — a reversal of ontogeny. Now both bile duct derived cells and hepatocytes can be isolated from the liver, genetically modified in vitro and returned to their in vivo origins where, after considerable population expansion, they can function as hepatocytes — paving the way for ex vivo gene therapy.     

Establishment of human parotid pleomorphic adenoma cells in culture: Morphological and biochemical characterization

Summary This study reports the establishment ofα-amylase-producing human parotid pleomorphic adenoma cell lines (2HP and 2HP₁) which have been maintained in culture for over 1 yr. The procedures required preparation of cellular clumps from tumor tissue and plating them on plasma clot or precoated dishes. During the initial phase of growth they required modified MCDB-153 medium without serum. When cells showed signs of degeneration they were changed to MCDB-153 medium containing first 2% and then 10% heat inactivated fetal bovine serum. Although cells grew well in MCDB-153 containing 10% serum, the epithelial cell morphology was not distinct. Therefore, the growth and morphology of cells grown in MCDB-10% serum were compared with those in RPMI growth medium containing 10% fetal bovine serum and F12 containing 10% agammaglobulin newborn bovine serum. Although the growth of cells was a little slower in F12 medium than those in MCDB and RPMI, the epithelial cell morphology was maintained better than in other growth media. The cells of 2HP and 2HP₁ produce low levels ofα-amylase and relatively high levels ofα-amylase mRNAs of 1176 and 702 bp and contain neurofilament-160, a neuronal-specific marker. The cells of 2HP₁ are tumorigenic when tested in athymic mice, but the cells of 2HP are not. The establishment of amylase-producing human parotid adenoma cell lines of different characteristics in culture provides a new opportunity to study the mechanisms of differentiation and transformation, and regulation ofα-amylase in these cells.