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J Lawry

Publications and source records attributed to J Lawry.

At least 19 recordsLinked to original sources
PubMed2003

Longitudinal study of chemokine receptor expression on peripheral lymphocytes in multiple sclerosis: CXCR3 upregulation is associated with relapse.

The interaction between chemokines and their receptors leads to selective recruitment of cells to foci of inflammation. Cross-sectional studies have reported significantly different expression of chemokine receptors CXCR3, CCR5 and CCR2 on peripheral blood lymphocytes in multiple sclerosis (MS) compared with controls. Cells expressing these receptors are likely to play a pathogenic role as suggested by studies of experimental autoimmune encephalomyelitis. Also, immunogenetic studies of nonfunctional CCR5 receptors in MS patients, due to 32delta deletion, demonstrated a delay in time to next relapse. The aims of this study were to detect any changes in the serial expression of chemokine receptors CCR2, CCR3, CCR5 and CXCR3 on peripheral blood CD4+ lymphocytes from patients with MS and to correlate the changes with relapses. Upregulation of CXCR3 expression on peripheral blood CD4+ lymphocytes was associated with all relapses and CCR5 expression was significantly affected with all relapses. Clinical recovery, with or without intravenous methylprednisolone treatment, coincided with the return of CXCR3 towards baseline in all but one case. Fluctuation in the expression of CXCR3 and CCR5 was also significantly greater in clinically stable patients with MS compared with controls, which may be due to subclinical disease activity. These findings provide further support for the view that CXCR3 and CCR5 antagonists could have a therapeutic value in MS.

Adult↗
PubMed2002

Monitoring tumour cells in the peripheral blood of small cell lung cancer patients.

BACKGROUND: Flow cytometry was used to enumerate tumour cells in longitudinal studies of peripheral blood from small cell lung cancer (SCLC) patients, together with magnetic bead selection to isolate and identify these cells. As part of a trial, 11 patients received either standard (four weekly) chemotherapy with ifosfamide, carboplatin, and etoposide (ICE) or accelerated (two weekly) ICE with filgrastim (granulocyte colony-stimulating factor [G-CSF]) and autologous stem cell support. METHODS: Fresh venous blood was taken throughout treatment and follow-up. Aliquots were stained with a "tumour-specific" antibody against epithelial tissue (Ber EP4), verified as a good marker of SCLC cells by immunohistochemistry. Matched samples labelled with Ber EP4 were separated magnetically by adding a secondary bead-antibody conjugate for confirmation of tumour cell identity. RESULTS: Circulating tumour cells were detected and monitored throughout treatment periods. An initial rise in circulating cells after the first cycle was followed by a fall in both treatment arms to baseline levels set by normal controls. This was achieved by week 12 in the accelerated treatment arm and by week 24 in the standard arm. CONCLUSIONS: Flow cytometry and magnetic bead isolation can be used to identify changes in numbers of circulating tumour cells in patients undergoing chemotherapy for SCLC and thereafter during follow-up periods. Absence of tumour cells may indicate a more favourable patient group who would benefit from a more intense course of treatment.

Antibody Affinity↗
PubMed2001

p53 and p21waf-1 expression correlates with apoptosis or cell survival in poorly differentiated, but not well-differentiated, retinoblastomas.

In human retinoblastomas, rare genetic mutations of the retinoblastoma gene cause massive cell proliferation, altered differentiation, and tumor formation; but paradoxically, this is accompanied by extensive apoptotic cell loss. We quantified the immunohistochemical distribution of p53, its downstream effector p21 (WAF-1), and apoptotic cells in 50 human retinoblastomas, within three concentric zones of sleeves of tumor cells surrounding blood vessels. In poorly differentiated retinoblastomas, both p53 expression and apoptosis increase toward the outer zone of tumor sleeves, whereas p21 expression occurs primarily within the inner zone. This staining pattern of p53 expression is reversed in well-differentiated tumors, whereas p21 staining and apoptotic cell distributions are unchanged. We detected no p53 mutations in four retinoblastomas and two retinoblastoma cell lines. We postulate that oxygen and cell "survival/growth factors" delivered via blood vessels protect retinoblastoma cells from apoptosis. In poorly differentiated tumors, apoptosis is spatially associated with increased p53 expression and may be p53 mediated, but in well-differentiated tumors, apoptosis does not colocalize with p53 and may be p53 independent. In retinoblastomas, p21 is involved not in cell death by apoptosis but in cell survival. Thus, p53 varies its expression (and by implication its function) with altered differentiation in retinoblastomas.

Apoptosis↗
PubMed2001

A study of death by anoikis in cultured epithelial cells.

BACKGROUND: Epithelial cells are critically dependent upon cell-matrix and cell-cell adhesion for growth and survival. Anoikis is programmed cell death caused by disruption of cell-substrate adhesion in normal epithelial cells. METHODS: We studied the induction of anoikis in vitro in two cell lines; HaCaT and SW742. PI3K, JAK2 and PKC are key elements in signalling pathways regulating cell survival, and using specific inhibitors we also examined their potential role in the induction of anoikis. RESULTS: When prevented from adhesion by culture on polyHEMA, HaCaT cells underwent apoptosis selectively from the proliferating population; surviving cells underwent cell cycle arrest. In SW742 cells anoikis also occurred, but was balanced by increased cycling. The effects of specific kinase inhibitors indicated that both Janus kinase 2 and protein kinase C partially protect HaCaT cells from anoikis through inducing cell cycle arrest of surviving nonadherent cells; inhibition of Phosphatidylinositol 3-kinase did not induce cycling in HaCaTs prevented from adhesion but did stimulate anoikis. SW742 cells showed markedly different responses: Janus kinase 2 inhibition activated apoptosis directly, Phosphatidylinositol 3-kinase inhibition stimulated both cell cycling and apoptosis, while protein kinase C inhibition stimulated cycling but inhibited apoptosis. CONCLUSIONS: Susceptibility to cell death in adhesion-prevented epithelial cells may thus be regulated by signalling pathways involving Phosphatidylinositol 3-kinase, Janus kinase 2 and protein kinase C. The ability of epithelial tumour cells to invade and metastasize may therefore result from disruption of these pathways.

Anoikis↗
PubMed2001

Over-diagnosis of hydatidiform mole in early tubal ectopic pregnancy.

AIMS: Tubal ectopic hydatidiform moles are rare lesions, and only 40 cases have been reported in the world literature. We investigated the apparently high incidence of tubal ectopic hydatidiform moles in women referred for treatment to a Supraregional Trophoblastic Tumour Screening and Treatment Centre between 1986 and 1996. METHODS AND RESULTS: Of 4261 women referred during the study period, 25 (0.6%) had a suspected tubal ectopic hydatidiform mole and paraffin-embedded tissue was available in 20 (80%) of these. Each case was reviewed by two pathologists and DNA flow cytometric analysis was undertaken when the histological diagnosis was initially deemed equivocal or suggestive of hydatidiform mole. On review, 17 cases (85%) showed no evidence of hydatidiform mole (circumferential trophoblastic proliferation, hydrops, scalloped villi, and stromal karyorrhexis). Of these, 11 cases (65%) showed features of early placentation and six (35%) showed hydropic abortion. DNA flow cytometry was performed in 14 (82%) of these cases and revealed a diploid population in each case. Three cases of molar pregnancy (15%) were identified. Each of these cases had the histological features of an early complete hydatidiform mole. Sufficient tissue was available for DNA flow cytometric analysis in two of these cases and confirmed the presence of diploidy in each. CONCLUSION: Our results show that tubal ectopic hydatidiform mole is a rare entity and demonstrate that it is over-diagnosed. Polar trophoblastic proliferation and hydropic villi are features of early placentation and of hydropic abortion. Sheets of extravillous trophoblast may be particularly prominent in tubal ectopic gestation. In the absence of circumferential trophoblastic proliferation combined with hydropic change a diagnosis of gestational trophoblastic disease should be avoided.

Abortion, Spontaneous↗
PubMed2000

Standardization of lymphocyte antibody binding capacity - a multi-centre study.

As quantitative flow cytometry is being increasingly used to characterize non-malignant and malignant disorders, interlaboratory standardization becomes an important issue. However, the lack of standardized methods and process controls with predefined antibody binding capacity values, limits direct interlaboratory comparison. The present study has addressed these issues using a stable whole blood product and a standardized antigen quantification protocol. It was demonstrated that: (i) a standard technical protocol can result in a high degree of interlaboratory concordance; (ii) interlaboratory variation of less than 12% can be achieved for CD4 antibody binding capacity values; and (iii) stable whole blood can be used as a process control with predefined antibody binding capacity values. Furthermore, using such an approach, a normal range was established for CD3, CD4 CD8 and CD19. These antigens appear to be expressed in a hierarchical manner, a factor that could be used as a procedural quality control measure.

Antigens, CD↗
PubMed1999

Stochastic acquisition of Qa1 receptors during the development of fetal NK cells in vitro accounts in part but not in whole for the ability of these cells to distinguish between class I-sufficient and class I-deficient targets.

Fetal mouse NK cells are grossly deficient in the expression of Ly49 molecules yet show a limited ability to distinguish between wild-type and MHC class I-deficient target cells. In this paper we report that during their development in vitro from immature thymic progenitors, a proportion of C57BL/6 fetal NK cells acquires receptors for a soluble form of the nonclassical class I molecule Qa1b associated with the Qdm peptide, but not for soluble forms of the classical class I molecules Kb and Db. The acquisition of these Qa1 receptors occurs in a stochastic manner that is strictly controlled by cytokines, and in particular is strongly inhibited by IL-4. All fetal NK clones tested, including those that lack detectable Qa1 receptors, express mRNA for CD94 and for both inhibitory and noninhibitory members of the NKG2 family. Fetal NK cells lacking receptors for Qa1 (and also for classical class I molecules) cannot distinguish between wild-type and class I-deficient blasts but, surprisingly, distinguish efficiently between certain wild-type and class I-deficient tumor cells. A variant line that lacks several members of the NKG2 family kills both types of tumor cell equally well, suggesting the existence of NKG2-containing inhibitory receptors that recognize as yet undefined nonclassical class I molecules of restricted distribution.

Animals↗
PubMed1999

The correlation between cell surface markers and clinical features in choroidal malignant melanomas.

PURPOSE: Uveal melanoma continues to present problems when attempting to predict disease progression. This study attempts to identify markers indicative of the biological characteristics of cells isolated from samples of uveal melanoma, including adhesion (ICAM-1), immune reactivity (MHC Class I and II), cell cycle control (c-erbB-2, c-myc) and apoptosis control (bcl-2, p53) using dual parameter (DNA/MoAb) flow cytometry. METHODS: Sixty-three fresh tissue samples from choroidal melanomas were taken at enucleation. Samples were assayed for DNA content and cell cycle, the above antibodies together with positive (PHM-5) and negative (2 degrees FITC Ab) controls. The clinical parameters sex, age, tumour location, cell type, tumour volume and presence of metastases were compared with the results and analysed with the non-parametric Mann-Whitney U-t-test. RESULTS: ICAM-1 expression proved to be the most clinically relevant, being present on a higher proportion of cells in tumours > 2000 mm3 (median 38, n = 19) compared with the smaller tumours < 2000 mm3 (median 17, n = 26) (p = 0.0015). Metastatic disease was present in 11 patients and did not correlate with any of the surface markers. C-myc, c-erbB-2 and MHC Class II expression were associated with cell type, all showing greater expression in spindle cell tumours than mixed/epithelial types. CONCLUSION: These results show flow cytometry as a quick, easy method to provide a 'phenotypic profile' for these tumours, and identifies cell cycle control and adhesion molecule expression as important areas for further investigation. c-erbB-2 and bcl-2 positivity was typically seen on over 60% cells in each sample, indicating two potential targets for therapeutic intervention.

Adult↗
PubMed1998

Cell cycle phase influences tumour cell sensitivity to aminolaevulinic acid-induced photodynamic therapy in vitro.

Photodynamic therapy (PDT) is a form of cancer treatment based on the destruction of cells by the interaction of light, oxygen and a photosensitizer. Aminolaevulinic acid (ALA) is the prodrug of the photosensitizer protoporphyrin IX (PpIX). ALA-induced PDT depends on the rate of cellular synthesis of PpIX, which may vary with cell cycle phase. This study has investigated the relationship between cell cycle phase, PpIX generation and phototoxicity in synchronized and unsynchronized bladder cancer cells (HT1197). In unsynchronized cells, relative PpIX fluorescence values (arbitrary units) were significantly different between cell cycle phases after a 1-h ALA incubation (G1 24.8 +/- 0.7; S-phase, 32.7 +/- 0.8, P < 0.05; G2 35.4 +/- 0.8, P < 0.05). In synchronized cells after a 1-h ALA incubation, cells in G1 produced less PpIX than those in S-phase or G2 [6.65 +/- 1.1 ng per 10(5) cells compared with 15.5 +/- 2.1 (P < 0.05), and 8.1 +/- 1.8 ng per 10(5) cells (not significant) respectively] and were significantly less sensitive to ALA-induced PDT (% survival, G1 76.2 +/- 8.3; S-phase 49.7 +/- 4.6, P < 0.05; G2 44.2 +/- 2.4, P < 0.05). This differential response in tumour cells may have implications for clinical PDT, resulting in treatment resistance and possible failure in complete tumour response.

Aminolevulinic Acid↗
PubMed1998

Simultaneous cell cycle and phenotypic analysis of primary uveal melanoma by flow cytometry.

BACKGROUND AND PURPOSE: DNA ploidy and cell cycle measurements of uveal melanoma tissue are regarded as having limited prognostic significance. In contrast, dual-parameter (DNA monoclonal antibody) flow cytometry offers a convenient and rapid way to screen tumour samples for a variety of phenotypic markers, whilst simultaneously measuring DNA ploidy and cell cycle, and therefore has the increased potential to identify clinically relevant indicators of disease progression. The aim of the present study was to identify a simple yet robust method for isolating, preserving and staining cells that could be analysed by flow cytometry. METHODS: Using a simple preparation procedure, a panel of membrane-associated antibodies (ICAM-1, W632, HLA-DR) and nuclear or cytoplasmic oncoprotein antibodies (c-erbB-2, c-myc, bcl-2, p53), together with positive (PHM-5) and negative (FITC F(ab')2) controls, were assayed. It was considered important to test the protocol with markers expressed on the cell surface, and in the cytoplasm and nucleus, so as not to be restrictive and thereby exclude an antigen of potential clinical interest. In addition, such panels would also enable the generation of a 'phenotypic profile' for each specimen that may reveal clinically significant trends. RESULTS: Our results indicate that tissue dissociation followed by brief fixation in 1% paraformaldehyde and permeabilisation in 70% methanol produces a stable single cell suspension, which can subsequently be stained with a wide range of antibodies for the accurate identification of cells in a potentially heterogeneous tumour population. CONCLUSION: This technology can rapidly identify sub-populations of cells expressing differing levels of proteins, which may prove to be indicative of disease progression for this aggressive disease.

Antibodies, Monoclonal↗
PubMed1998

Interleukin-6 is expressed by plasma cells from patients with multiple myeloma and monoclonal gammopathy of undetermined significance.

Interleukin-6 (IL-6) is an important growth factor for human myeloma cells in vitro and in vivo. However, the identity of the cells producing IL-6 in vivo in patients with multiple myeloma (MM) remains the subject of debate. We have developed a sensitive dual-colour fluorescence in situ hybridization (FISH) technique to investigate the expression of IL-6 mRNA by individual bone marrow plasma cells from patients with multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS) and healthy subjects. IL-6 mRNA could be identified in all immunoglobulin light chain (IgLC) expressing cells from all patients with MM and MGUS. The IL-6 protein could also be detected by direct immunofluorescence in all plasma cells (cytoplasmic light chain positive) from all patients with MM and MGUS. Furthermore, it was also possible to demonstrate cytoplasmic IL-6 staining of plasma cells from patients with MM by flow cytometric analysis. In contrast, neither the IL-6 mRNA or protein could be detected in normal plasma cells from healthy bone marrow donors. These data demonstrate that plasma cells from patients with MM and MGUS express the IL-6 mRNA and synthesize the IL-6 protein and support the hypothesis that autocrine synthesis of IL-6 is of importance in patients with MM.

Aged↗
PubMed1998

Human myeloma cells shed the interleukin-6 receptor: inhibition by tissue inhibitor of metalloproteinase-3 and a hydroxamate-based metalloproteinase inhibitor.

Interleukin-6 (IL-6) is the major growth factor for human myeloma cells, exerting its effect through the IL-6 receptor (IL-6R). A soluble form of IL-6R (sIL-6R) has been identified, which increases the sensitivity of myeloma cells to IL-6. In patients with multiple myeloma (MM), serum concentrations of sIL-6R are elevated and associated with poor prognosis. The present study was undertaken to determine whether proteolytic cleavage of IL-6R could contribute to sIL-6R release from human myeloma cells, and also to identify the class of proteinase responsible for this event. Human myeloma cell lines were shown to express IL-6R upon their surface and also to release sIL-6R into culture supernatants. In addition, phorbol 12-myristate 13-acetate (PMA) stimulated a loss of IL-6R from the cell surface, with a corresponding increase in the concentration of sIL-6R in the supernatant. Inhibitors of serine and cysteine proteinases, and tissue inhibitor of metalloproteinase (TIMP) -1 and TIMP-2, were shown to have no effect on the magnitude of sIL-6R release. In contrast, TIMP-3 and a hydroxamate-based metalloproteinase inhibitor (BB-94), inhibited both constitutive and PMA-induced release of sIL-6R. Myeloma cells freshly isolated from the bone marrow of a patient with MM were also shown to express IL-6R upon their surface, and to shed this receptor in response to PMA. These data demonstrate that increased proteolytic cleavage of IL-6R, mediated by a non-matrix-type metalloproteinase, is likely to contribute to the elevated concentrations of sIL-6R found in the serum of patients with MM. Inhibition of sIL-6R release by hydroxamate-based metalloproteinase inhibitors may represent a novel therapeutic approach to the treatment of MM.

Bone Marrow Cells↗
PubMed1997

The cell cycle and induction of apoptosis in a hamster fibrosarcoma cell line treated with anti-cancer drugs: its importance to solid tumour chemotherapy.

The induction of apoptosis by anticancer drugs and its relationship to stages of the cell cycle was studied in cells derived from a solid tumour; a highly malignant hamster fibrosarcoma (Met B). Asynchronously proliferating cells were treated with a wide variety of agents such as actinomycin-D, 1-beta-D-arabinofuranosyl cytosine, camptothecin, cisplatin, cyclophosphamide, daunorubicin, 5-flurouracil, 6-mercaptopurine, hydroxyurea, ionomycin, methotrexate and vincristine. With the exception of cyclophosphamide and hydroxyurea, a 36 h exposure to these drugs resulted in inhibition of cell growth and apart from cyclophosphamide, hydroxyurea. 6-mercaptopurine and cisplatin the induction of apoptosis. Studies using a decreased concentration of drug and exposure time (12 h) followed by examination of cells using flow cytometry indicated that most drugs were capable of affecting cell cycle progression without induction of apoptosis. However when cells were synchronised at G0/G1, S and G2/M phases and then exposed to these decreased concentrations of drug apart from 6MP an HU, apoptosis was observed and for the majority of drugs it took place in the same phase in which progression through the cell cycle was blocked by the drug. Cells synchronised in G0/G1 phase were more susceptible to methotrexate, whereas S-phase cells were more susceptible to camptothecin and 5-flurouracil and G2/M phase cells more susceptible to actinomycin D, 1-beta-D-arabinofuranosyl cytosine, daunorubicin and cisplatin. In contrast, vincristine blocked cells in G2/M phase but exerted its apoptotic effect in S-phase cells, ionomycin had no effect on the cell cycle, but G2/M cells appeared to be more susceptible to the effect of this drug. These data indicate that entry into apoptosis by this fibrosarcoma may occur at any point in the cell cycle. They also demonstrate a correlation between the action of some anticancer drugs on the cell cycle and the subsequent induction of apoptosis which may be useful in chemotherapeutic design.

Animals↗
PubMed1997

Cytokine modulation of antigen expression in human melanoma cell lines derived from primary and metastatic tumour tissues.

The constitutive and cytokine-mediated expression of MHC class I and II antigens and intercellular adhesion molecule-1 (ICAM-1) was evaluated on eight human melanoma cell lines derived from primary and metastatic malignancies from patients (WM human melanoma series) including three pairs of related cell lines derived from the same individual. The cytokines IL-1 beta, IL-4, IL-6, TNF alpha, TGF beta 2, IFN gamma and IFN alpha were assessed for their ability to modulate the expression of cell surface antigens. MHC class I and class II antigen expression was unregulated by IFN gamma, IFN alpha and/or TNF alpha in cell lines established from primary melanoma. In contrast the cell lines derived from metastatic deposits did not show an increase in expression of MHC antigens in response to these cytokines. Both primary and metastatic WM cell lines were shown to be resistant to spontaneous natural killer cell (NK) activity, but susceptible to effector lymphocytes mediating lymphotine activated killer (LAK) cytotoxicity as a result of activation by IL-2. Although the constitutive and cytokine-induced level of expression of ICAM-1 and MHC antigens varied between paired primary and metastatic cell lines, this did not correlate with susceptibility of the cell line target to NK or LAK cytotoxicity. Whereas the IFNs, TNF alpha, TGF beta 2 and IL-1 beta differentially modulated the expression of ICAM-1 and MHC class I, treatment with IFNs (but not IL-1 beta, TNF alpha or TGF beta 2) resulted in a significant reduction in the sensitivity of the melanoma cells to NK and LAK cytotoxicity. Constitutive ICAM-1 expression was positively correlated with the ability of WM cell lines to colonise the lungs of SCID mice upon i.v. injection. The acquisition of cytokine resistance and inability to demonstrate enhanced cell surface expression may represent an important feature associated with the development of the metastatic phenotype.

Animals↗
PubMed1996

Bisphosphonates induce apoptosis in mouse macrophage-like cells in vitro by a nitric oxide-independent mechanism.

Bisphosphonates (BPs) are an important class of antiresorptive drugs used in the treatment of bone diseases, including osteoporosis. Although their mechanism of action has not been identified at the molecular level, there is substantial evidence that BPs can have a direct effect on osteoclasts by mechanisms that may lead to osteoclast cell death by apoptosis. BPs can also inhibit proliferation and cause cell death in macrophages in vitro. We have now shown that the toxic effect of BPs on macrophages is also due to the induction of apoptotic, rather than necrotic, cell death. Morphological and biochemical features that are definitive of apoptosis (chromatin condensation, nuclear fragmentation, and endonuclease-mediated internucleosomal cleavage of DNA) could be identified in mouse macrophage-like J774 and RAW264 cells, following treatment with 100 microM pamidronate, alendronate, and ibandronate for 24 h or more. Clodronate was much less potent, even at 2000 microM, while 2000 microM etidronate did not cause apoptosis. Apoptosis was not due to increased synthesis of nitric oxide and could not be prevented by inhibitors of nitric oxide synthases. Since macrophages, like osteoclasts, are particularly susceptible to BPs, these observations support the recent suggestion that the mechanism by which BPs inhibit bone resorption may involve osteoclast apoptosis. Furthermore, the macrophage-like cell lines used in this study may be a convenient model with which to identify the molecular mechanisms by which BPs promote apoptosis in osteoclasts. Induction of macrophage apoptosis by BPs in vivo may also account, at least in part, for the anti-inflammatory properties of BPs as well as the ability of BPs to cause an acute phase response.

Alendronate↗
PubMed1996

Detection of circulating prostate-specific antigen-positive cells in patients with prostate cancer by flow cytometry and reverse transcription polymerase chain reaction.

The presence of prostate-specific antigen (PSA)-positive cells has previously been demonstrated in the peripheral blood of prostate cancer patients by flow cytometry (FC), but the identity of these cells has not been established. In this study, the reverse transcriptase polymerase chain reaction (RT-PCR) was compared with analytical FC in an attempt to detect and characterise these cells. Peripheral blood was obtained from 12 patients with newly diagnosed and untreated prostate cancer and five controls. Nine of the 12 patients with prostate cancer (75%) had circulating PSA-positive cells as shown by FC. Only one of those patients (11.1%) was found to express PSA mRNA by RT-PCR. The absence of PSA mRNA in the majority of samples showing PSA-positive cells suggests that they do not represent haematogenous micrometastases. PSA-positive cells in the blood could represent monocytes that express PSA, either following binding/phagocytosis of free serum PSA or phagocytosis of tumour cells.

Base Sequence↗
PubMed1996

Measurement of intracellular pH in human spermatozoa by flow cytometry with the benzo[c]xanthene dye SNAFL-1: a novel, single excitation, dual emission, molecular probe.

In numerous animal species the acrosome reaction of spermatozoa has been linked to elevations in intracellular pH (pHi). However, whether or not this is merely a passive consequence of calcium ion influx is not known. Studies into the fluctuations in pHi in sperm cells have been hampered by the lack of a pH-sensitive probe that could be used in conjunction with flow cytometry. In this study, flow cytometric analysis of pHi in human spermatozoa was accomplished by using one of the new benzo[c]xanthene dyes (SNAFL-1). SNAFL-1 was then observed in situ with conventional fluorescent microscopy and was found to be located in the post-acrosomal cytoplasm of the head. It was then used to measure the differences in pHi between acrosome intact populations of spermatozoa, and populations that had been induced to acrosome-react with human follicular fluid or the calcium ionophore A23187 to mimic the calcium influx. It was concluded that the human sperm acrosome reaction is also accompanied by a rise in pHi and the natural agonist-induced rise could not be accounted for by calcium ion influx alone.

Acrosome↗