Thiruchandurai V. Rajan

Mutations at the murine motheaten locus are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene

Mice homozygous for the recessive allelic mutation motheaten (me) or viable motheaten (mev) on chromosome 6 develop severe defects in hematopoiesis. In this paper we present the findings that the me and mev mutations are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene. High resolution mapping localized me to an area tightly linked to Hcph on chromosome 6. Abnormalities of the Hcph protein product were demonstrated by Western blot analysis and by activity assays in both me/me and mev/mev mice. Molecular analysis of the Hcph cDNA identified abnormal transcripts in both mutants. DNA sequence analyses of cDNA and genomic clones revealed that both the me and mev mutations are point mutations that result in aberrant splicing of the Hcph transcript. These findings provide the first available animal models for a specific protein-tyrosine phosphatase deficiency, thus facilitating determination of the precise role of this signaling molecule in hematopoiesis.

Natural but Not Inducible Regulatory T Cells Require TNF-α Signaling for In Vivo Function

TNF-α has a multifunctional role in autoimmune diseases as reflected in the variable responses of different human diseases to anti–TNF-α therapy. Recent studies have suggested that TNF-α modulates autoimmunity partially via effects on regulatory T cells (Tregs) and that these effects are mediated through the type II TNFR (TNFR2). We have investigated the requirement for TNFR2-expression on murine natural Tregs (nTregs) and induced Tregs (iTregs) in mediating suppression of colitis. Surprisingly, we find that TNFR2-expression is required for both spleen- and thymus-derived nTreg-mediated suppression, but is not required for iTreg-mediated suppression. Abnormal TNFR2−/− nTreg function was not associated with an in vivo decrease in accumulation, stability, or expression of markers known to be relevant in Treg function. Because iTregs are generated in the presence of TGF-β, we investigated whether activation in the presence of TGF-β could overcome the functional defect in TNFR2−/− nTregs. Although preactivation alone did not restore suppressive function of nTregs, preactivation in the presence of TGF-β did. These results identify potentially critical differences in activation requirements for nTregs versus iTregs. Furthermore, our findings are consistent with reports suggesting that nTregs are activated in sites of inflammation while iTregs are activated in lymph nodes. Finally, by demonstrating that nTregs require TNF-α for optimal function whereas iTregs do not, our results suggest that the enigma of variable responses of different human diseases to anti–TNF-α therapy may relate to whether nTregs or iTregs have the predominant regulatory role in a given disease.

Role of gamma interferon and interleukin-4 in host defense against the human filarial parasite Brugia malayi.

ABSTRACT We have investigated the roles of gamma interferon (IFN-γ) and interleukin-4 (IL-4) in host defense against Brugia malayi. Our data suggest that the lack of either IFN-γ or IL-4 prolongs the time required to achieve sterile immunity, suggesting that both canonical type 1 and type 2 responses are involved in the clearance of infection.

Critical Role for IgM in Host Protection in Experimental Filarial Infection

We have previously shown that B cells (in particular B1 cells) are important in host protection against brugian infections in a murine i.p. model. In this study, we show that mice deficient in circulating IgM (secIgM−/−), but otherwise normal in their humoral responses, manifest a significant impairment in worm elimination, suggesting that one critical B cell function is the production of Ag-specific IgM. Efficient elimination of larvae is IgM dependent for both primary and challenge infections. The ability to eliminate worms is restored in secIgM−/− mice by administering sera from primed mice. We corroborated these in vivo studies with in vitro observations which show that IgM is the only isotype that reacts strongly with the surface of Brugia L3. Furthermore, activated peritoneal exudate cells adhere to L3 only in the presence of filaria-specific sera or IgM purified from them. This attachment is not reduced by heat inactivation of the serum, suggesting complement independent activity. Peritoneal exudate cells from primed mice, especially activated macrophages, carry high levels of IgM on their surfaces. Our observations suggest that an IgM-mediated reaction initiates the formation of host-protective granulomas.

Brugian infections in the peritoneal cavities of laboratory mice: kinetics of infection and cellular responses.

Standard, immunocompetent, inbred strains of mice are non-permissive for infection with the human filarial nematode, Brugia malayi or the closely related Brugia pahangi. This non-permissiveness allows one to address the mechanism(s) that might be used by mammalian hosts to eliminate large, multicellular, metazoan, extracellular invertebrate pathogens. We describe here the time course of intraperitoneal Brugian infections in naïve and primed +/+ mice from two commonly used, inbred laboratory strains (C57BL/6J and BALB/cByJ). We believe that this documentation of the course of infection in normal mice will serve as a reference for future studies using mice with gene-targeted immunological deficits or which have been pharmacologically or immunologically manipulated to manifest such deficits. Our data show that even though both strains of mice eliminate the parasite before the onset of patency, there are significant differences in the time course of infection and in the fractions of input larvae that can be recovered at any time after infection. In a secondary infection, the time course of elimination is accelerated. We examined the cells in the peritoneal cavity, the site of infection, by flow microfluorimetry using forward and side scatter properties and cell surface antigen expression using fluorescent antibodies. These studies reveal a complex cellular pattern, predominated by B lymphocytes, macrophages, and eosinophils. The most notable gross morphological findings at necropsy during the phase of elimination of the parasite are nodules of tissue containing larvae, which appear viable in some cases and undergoing various stages of disintegration in others. These nodules, which are histologically granulomas, are primarily composed of macrophages and eosinophils, with few if any lymphocytes. Transmission electron micrographs reveal that eosinophils can penetrate under the cuticles of the larvae and be seen in close approximation with internal structures. These granulomas may represent an important mechanism by which worms are eliminated.

Immune responses to third stage larvae of Onchocerca volvulus in interferon‐gamma and interleukin‐4 knockout mice

To shed clarity on the dichotomy of reported results relative to the significance of T helper‐1 vs T helper‐2 immune responses in onchocerciasis, we compared the survivability of Onchocerca volvulus third‐stage larvae (L3) in immunized mice that had either a targeted disruption of the Interleukin‐4 or Interferon‐gamma gene. Treatment groups consisted of control mice and mice immunized with irradiated O. volvulus L3. All mice were challenged with diffusion chambers containing viable L3. Vaccinated IL‐4−/− were unable to kill this larval target. In contrast, vaccinated IFN‐γ−/− and C57BL/6 mice, exhibited high levels of killing, had elevated levels of IL‐4 and significantly greater numbers of eosinophils in their diffusion chambers than the IL‐4−/−. Whereas, levels of IFN‐γ in all three groups of immunized mice were equivalent to those of control mice, levels of IL‐5 were elevated, even in the IL‐4−/−, indicating that cytokines other than IL‐4 were involved in its production. The protective immune response to third‐stage larvae of O. volvulus in mice vaccinated with irradiated larvae has an absolute IL‐4 requirement.

Expression of secretory phospholipase A2 in colon tumor cells potentiates tumor growth.

Secretory phospholipase A2 (sPLA2‐IIA) has been shown to attenuate intestinal tumorigenesis in ApcMin mice, demonstrating that it is a tumor modifier. To further explore the actions of sPLA2‐IIA in tumorigenesis, sPLA2‐IIA was overexpressed in two cell lines where it is normally absent, the murine colon tumor cell line AJ02nm0, and human colon carcinoma cell line HCT‐116. Two allelic variants of sPLA2‐IIA were tested in this study; sPLA2‐IIAAKR and sPLA2‐IIASWR, which are derived from AKR/J and SWR/J mice, respectively, and differ by a single amino acid at position 63 in the calcium‐ and receptor‐binding domain. There was no change in cell‐doubling time for either allele when compared to vector controls. Furthermore, sodium butyrate and arachidonic acid (AA)‐induced cell death were unchanged in control and transfected cells. Addition of the sPLA2 substrate, palmitoyl‐arachidonoyl‐phosphatidic acid (PAPA), to AJ02nm0 cells resulted in a modest (12%–24%), but significant (P < 0.01), inhibition of growth that was dependent on sPLA2‐IIA expression. However, when AJ02nm0 and HCT‐116 cells were injected subcutaneously (sc) into nude mice, Pla2g2a expression resulted in a 2.5‐fold increase in tumor size. In addition, sPLA2‐IIA expressing HCT‐116 tumors were found to be more infiltrative than controls. We conclude that the ability of sPLA2‐IIA to slow tumor cell growth is dependent upon the availability of substrate, and that in some instances sPLA2‐IIA may actually enhance tumor growth. Mechanisms that may account for differences between the tumor explant model versus the ApcMin model of intestinal cancer are discussed.

Characterization of an hsp70 gene from the human filarial parasite, Brugia malayi (Nematoda).

We have previously shown that an Onchocerca volvulus cDNA clone in lambda gt-11 designated OvG15, potentially encoding a peptide homologous to the 70-kDa heat shock protein (Hsp70), was recognized by sera of many individuals living in a zone endemic for lymphatic filariasis and most strikingly by sera from amicrofilaremic individuals including endemic normals, those with chronic symptoms and TPE patients. Few asymptomatic microfilaremics recognized the Hsp70. We have now used the insert from the OvG15 clone to isolate the homologous gene from Brugia malayi and analyze its primary structure and expression. The data presented in this communication describe a heat-inducible member of the hsp70 gene family of B. malayi which demonstrates intriguing features of tissue specific basal level expression, developmental regulation and heat inducibility.

Interleukin-4 receptor-Stat6 signaling in murine infections with a tissue-dwelling nematode parasite.

ABSTRACT Interleukin-4 (IL-4) has been shown to be crucial in parasite expulsion in several gastrointestinal nematode infection models. Data from both epidemiological studies with humans and experimental infections in animals imply a critical role for the type II helper response, dominated by IL-4, in host protection. Here we utilized inbred mice on two distinct backgrounds to document the involvement of IL-4 in the clearance of a primary infection of Brugiafrom the murine host. Our data from infections of IL-4 receptor−/− and Stat6−/− mice further indicate that IL-4 exerts its effects by activating the Stat6 molecule in host target cells, a finding which links clearance requirements of a gastrointestinal tract-dwelling nematode with those of a tissue-dwelling nematode. Additionally, we show that the requirements for IL-4 receptor binding and Stat6 activation extend to accelerated clearance of a secondary infection as well. The data shown here, including analysis of cell populations at the site of infection and infection of immunoglobulin E (IgE)−/− mice, lead us to suggest that deficiencies in eosinophil recruitment and isotype switching to IgE production may be at least partially responsible for slower parasite clearance in the absence of IL-4.

Increased Frequency of Serrated Aberrant Crypt Foci Among Smokers

OBJECTIVES:The American College of Gastroenterology has published guidelines recently that suggest that smokers with a history of >20 pack years may need screening for colorectal cancer (CRC) at an earlier age than non-smokers. Aberrant crypt foci (ACF) may represent important precursors for colorectal neoplasms and potential surrogate biomarkers. Clarifying the role of ACF in relation to known CRC risk factors such as smoking may have important implications for screening as well as our understanding of tobacco use and colorectal carcinogenesis. Our goal was to examine whether smoking at least 20 pack years was associated with an increased frequency of ACF.METHODS:We gathered detailed smoking history, personal and family history of CRC, and other epidemiologic data (age, gender, height, weight, ethnicity, and medication use) from 125 patients undergoing routine screening or surveillance colonoscopy. We used a magnifying colonoscope (Olympus Close Focus Colonoscope XCF-Q160ALE, Olympus Corporation, Tokyo, Japan) and examined the distal 20 cm section of colon after staining with 0.5% methylene blue. ACF were counted and characterized histologically. Hyperplastic ACF were further characterized as either serrated or non-serrated.RESULTS:Smoking at least 20 pack years was associated with an increased likelihood (adjusted odds ratio (OR)=3.45; 95% confidence interval (CI)=1.93–6.18) of having more than the median number of ACF (≥15) compared with non-smokers. Similarly, patients with a personal history of advanced neoplasia were more likely (adjusted OR=3.42; 95% CI=1.01–11.67) to have a greater than median number of ACF compared with patients without this diagnosis. Smokers were more likely than non-smokers to have serrated ACF (P=0.002).CONCLUSIONS:Smoking at least 20 pack years seems to be associated with increased number of ACF in the rectum and distal sigmoid, especially those with serrated histology. Our data support ACG guidelines for earlier screening for CRC among smokers and add to our understanding of how colorectal carcinogenesis is related to tobacco use.